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Biomimetic Synthesis of Hierarchically Porous Nanostructured Metal Oxide Microparticles;Potential Scaffolds for Drug Delivery and Catalysis )
Gulaim A. Seisenbaeva,*,† Micheal P. Moloney,‡ Renata Tekoriute,‡ Adeline Hardy-Dessources,§, Jean-Marie Nedelec,§, Yurii K. Gun’ko,‡ and Vadim G. Kessler*,†
Department of Chemistry, SLU, P.O. Box 7015, 750 07 Uppsala, Sweden, ‡The School of Chemistry and CRANN Institute, Trinity College Dublin, Dublin 2, Ireland, §Clermont Universit e, ENSCCF, Laboratoire des Mat eriaux Inorganiques, BP 10448, F-63000 Clermont-Ferrand, France, and CNRS, UMR 6002, LMI, F-63177 Aubi ere, France )
†
Received January 7, 2010. Revised Manuscript Received March 3, 2010 Hierarchically porous hybrid microparticles, strikingly reminscent in their structure of the silica skeletons of singlecell algae, diatoms, but composed of titanium dioxide, and the chemically bound amphiphilic amino acids or small proteins can be prepared by a simple one-step biomimetic procedure, using hydrolysis of titanium alkoxides modified by these ligands. The growth of the hierarchical structure results from the conditions mimicking the growth of skeletons in real diatoms;the self-assembly of hydrolysis-generated titanium dioxide nanoparticles, templated by the microemulsion, originating from mixing the hydrocarbon solvent and water on action of amino acids as surfactants. The obtained microsize nanoparticle aggregates possess remarkable chemical and thermal stability and are promising substrates for applications in drug delivery and catalysis. They can be provided with pronounced surface chirality through application of chiral modifying ligands. They display also high selectivity in sorption of phosphorylated biomolecules or medicines as demonstrated by 1H and 31P NMR studies and by in vitro modeling using 32P-marked ATP as a substrate. The release of the adsorbed model compounds in an inert medium is a very slow process directed by desorption kinetics. It is enhanced, however, noticeably in contact with biological fluids modeling those of the tissues suffering inflammation, which makes the produced material highly attractive for application in medical implants. The developed synthetic approach has been applied successfully also for the preparation of analogous hybrid microparticles based on zirconium dioxide or aluminum sesquioxide.
Introduction The interest in oxide materials with well-developed pore structure and combination of high pore volume with high specific surface is caused by a huge number of possible applications for such objects varying from heterogeneous catalysts1 and catalyst supports2 to coatings on bone implants3 and potential carriers in drug delivery.4-6 The approaches to zeolite type materials7 and mesoporous silicas8 are well-developed and involve application of bulky9 or even polymer surfactants (especially amphiphilic block-copolymers such as pluronic 123)10 that offer well-defined open porosity. Considerable success has even been achieved recently in design and synthesis of chiral alumosilicate11 and even (1) Shiju, R. N.; Guliants, V. V. Appl. Catal., A 2009, 356, 1–17. (2) Feng, H.; Elam, J. W.; Libera, J. A.; Pellin, M. J.; Stair, P. C. Chem. Eng. Sci. 2009, 64, 560–567. (3) Wohlfahrt, J. C.; Monjo, M.; Ronold, H. J.; Aass, A. M.; Ellingsen, J. E.; Lyngstadaas, S. P. Clin. Oral Implants Res. 2010, 21, 165–173. (4) Slowing, I. I.; Trewyn, B. G.; Giri, S.; Lin, V. S.-Y. Adv. Funct. Mater. 2007, 17, 1225–1236. (5) Trewyn, B. G.; Giri, S.; Slowing, I.; Lin, V. S.-Y. Chem. Commun. 2007, 3236–3245. (6) Vallet-Regi, M.; Balas, F.; Arcos, D. Angew. Chem., Int. Ed. 2007, 46, 7548– 7558. (7) Perez-Ramirez, J.; Christensen, C. H.; Egeblad, K.; Christensen, C. H.; Groen, J. C. Chem. Soc. Rev. 2008, 37, 2530–2542. (8) Kresge, C. T.; Leonowicz, M. E.; Roth, W. J.; Vartuli, J. C.; Beck, J. S. Nature 1992, 359, 710–712. (9) Sakthivel, A.; Komura, K.; Huang, S. J.; Wu, P. H.; Liu, S. B.; Sasaki, Y.; Sugi, Y. Ind. Eng. Chem. Res. 2010, 49, 65–71. (10) Smarsly, B.; Antonietti, M. Eur. J. Inorg. Chem. 2006, 1111–1119. (11) Dryzun, C.; Mastai, Y.; Shvalb, A.; Avnir, D. J. Mater. Chem. 2009, 19, 2062–2069. (12) Sun, J. L.; Bonneau, C.; Cantin, A.; Corma, A.; Diaz-Cabanas, M. J.; Moliner, M.; Zhang, D.; Li, M.; Zou, X. D. Nature 2009, 458, 1154–U90.
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germanosilicate12 zeolite type structures. Nature itself has also developed approaches to this attractive type of material. Mineral skeletons of single-cell organisms such as radiolarians and diatoms are highly symmetric and highly porous nanomaterial constructions fascinating both biologists and specialists in materials synthesis with their complexity and functionality.13 They have already found broad applications within such domains as catalysis (as highly porous thermally stable supports),14 bioactive coatings,15 and sensor materials.16 Diatom skeleton is formed via biomineralization. Diatoms create a colloid double-phase oil (membrane lipid)-water system, stabilized by protein surfactants, from which a silica polymer is precipitating.17 Proteins have even been shown to enhance the condensation of silica.18 Microbial skeletons formed solely of metal oxides are unknown. Highly porous metal oxides, however, are extremely attractive as catalyst supports19 and as components of bone implants and potential sources for drug delivery20 in view of their ability to selective chemisorption of organic compounds via complexation on the surface. Recent studies have even revealed potential of titania nanotubes as containers for delivery of small molecule and (13) Sanchez, C.; Arribart, H.; Guille, M. M. G. Nat. Mater. 2005, 4, 277–288. (14) Jia, Y.; Han, W.; Xiong, G.; Yang, W. Sci. Technol. Adv. Mater. 2007, 8, 106–109. (15) Lopez-Alvarez, M.; Solla, E. L.; Gonzalez, P.; Serra, J.; Leon, B.; Marques, A. P.; Reis, R. L. J. Mater. Sci. Mater. Med. 2009, 20, 1131–1136. (16) Gale, D. K.; Gutu, T.; Jiao, J.; Chang, C. H.; Rorrer, G. L. Adv. Funct. Mater. 2009, 19, 926–933. (17) B€auerlein, E. Angew. Chem., Int. Ed. 2003, 42, 614–641. (18) Pohnert, G. Angew. Chem., Int. Ed. 2002, 41, 3167–3171. (19) Hakim, S. H.; Shanks, B. H. Chem. Mater. 2009, 21, 2027–2038. (20) Wu, J.-M.; Hayakawa, S.; Tsuru, K.; Osaka, A. J. Am. Ceram. Soc. 2004, 87, 1635–1642.
Published on Web 03/15/2010
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macromolecular drugs.21 Fully crystalline titanium oxide nanoparticles conjugated with immunoglobulin were found to be interesting as potential drug delivery vectors, especially in combination with their potential photochemically initiated cytotoxicity.22 Bulk hierarchically porous mesoporous-macroporous metal oxides have been produced recently via spontaneous hydrolysis of liquid metal-organic precursors, metal alkoxides, in water,19,23,24 often applying surfactants to maintain the desired porosity. Preparation of hierarchically porous titania films has been achieved only with application of block copolymers as surfactants and of complex solvent mixtures.25 A common feature of the thus-produced materials is that they consist of roughly shaped pieces of dry gels with smooth surface and sharp edges,19 most often with closed porosity, which leaves the available active surface strongly variable and can set hinders for biomedical applications. Synthesis of hierarchically porous silica particles has been successfully achieved recently either via application of a combination of surfactants26 or through enhancement of the reactivity of silica precursors via their modification with glycolate ligands and adding additionally a surfactant.27 We have shown here that metal oxide nanoparticle assemblies with controlled size and shape strikingly reminding diatoms, and possessing an open hierarchically porous structure, can be synthesized in the complete absence of artificial surfactants. This approach is mimicking that used by nature itself for the synthesis of silica skeletons in real diatoms and is exploiting self-assembly in the formation of metal oxide nanoparticles in combination with surfactant action of amphiphilic amino acids as modifying ligands.
Experimental Section All operations with the synthesis and handling of precursors, and their solutions have been carried out applying dry nitrogen atmosphere in a glovebox. Hydrocarbon solvents, toluene, pentanes, and n-hexane were purchased from Aldrich and purified by refluxing over LiAlH4 with subsequent distillation. Alcohols, n-propanol, and isopropanol were purchased from Merck and purified by refluxing over corresponding aluminum alkoxides with subsequent distillation. The metal alkoxides used in this work, Ti(OiPr)4, (TiOnPr)4, Zr(OnPr)4 70 wt % solution in n PrOH, and Al(OiPr)3, as well as the modifying ligands, L-Penicillamine, D-Penicillamine, ethylphosphonic acid, 2-aminoethyl phosphonic acid, and disodium salt of adenosine triphosphonic acid (ATP), were purchased from Aldrich and used without further purification. 32P-marked ATP (substitution on γ-phosphorus atom, 1 mL of solution with 1 mCurie total β-activity) was purchased from Perkin-Elmer and handled by the LigandTracer Inc. at the certified facility located at the Rudbeck Laboratory in Uppsala, Sweden). IR spectra of nujol mulls were registered with a Perkin-Elmer FT-IR spectrum 100 spectrometer. NMR spectra were recorded for the precursors as CDCl3 solutions and for the nanoparticles as D2O solutions on a Bruker 400 MHz spectrometer at 303 K. The 31 P spectra were run for the reference solution of aminoethylphosphonic acid in D2O and also for the suspension of 30 μm particles (21) Peng, L.; Mendelsohn, A. D.; LaTempa, T. J.; Yoriya, S.; Grimes, C. A.; Desai, T. A. Nano Lett. 2009, 9, 1932–1936. (22) Rozhkova, E. A.; Ulasov, I.; Lai, B.; Dimitrijevic, N. M.; Lesniak, M. S.; Rajh, T. Nano Lett. 2009, 9, 3337–3342. (23) Yuan, Z.-Y.; Vantomme, A.; Leonard, A.; Su, B.-L. Chem. Commun. 2003, 1558–1559. (24) Blin, J.-L.; Leonard, A.; Yuan, Z.-Y.; Gigot, L.; Vantomme, A.; Cheetham, A. K.; Su, B.-L. Angew. Chem., Int. Ed. 2003, 42, 2872–2875. (25) Malfatti, L.; Bellino, M. G.; Innocenzi, P.; Soler-Illia, G. J. A. A. Chem. Mater. 2009, 21, 2763–2769. (26) Carroll, N. J.; Pylypenko, S.; Atanassov, P. B.; Petsev, D. N. Langmuir 2009, 25, 13540–13544. (27) Schiller, R.; Weiss, C. K.; Geserick, J.; Husing, N.; Landfester, K. Chem. Mater. 2009, 21, 5088–5098.
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stored under 15 mM solution of aminoethylphosphonic acid for 1 week, separated by decantation, rinsed in D2O, and then redispersed in D2O. The X-ray single crystal and powder studies have been carried out with a multipurpose Bruker SMART CCD 1K diffractometer using Mo KR radiation (0.710 73 A˚). SMART software was used for the data collection and SAINTPLUS or APEX-2 program packages for the data reduction and integration for the single crystal and powder experiments, respectively. AFM images were recorded using Bruker NANOS AFM/SPM microscope at the Bruker NANO research facility in Karlsruhe, Germany. SEMEDS studies were carried out with a Hitachi TM-1000-μDeX scanning electron microscope. TEM images were taken on a JEOL JEM-2100 LaB6 operating at 200 kV on samples prepared by deposition and drying of a drop of an aqueous suspension onto a Formvar-coated 400 mesh copper grid. Thermogravimetric characterization of the samples was carried out with a PerkinElmer Pyris 1 TGA instrument. Elementary microanalyses were carried out by MikroKemi AB, Uppsala, Sweden. Synthesis of Precursor Compounds. In a typical procedure, 1 mL of liquid titanium alkoxide was dissolved in 5 mL of toluene, and to the obtained solution, 1 equiv (about 0.63 g) of fine powder of Penicillamine was added on vigorous shaking. The mixture was subjected to reflux until complete dissolution of the ligand, and the resulting yellow solution was left in a freezer overnight for crystallization. The light yellow to orange crystals were separated by decantation and dried in vacuum. Typical yields were 1.1-1.2 g (76-82%). Found: C 44.8-46.0; H 7.9-8.3; N 3.8-4.0; S 8.4-8.7. Calculated for Ti(OC3H7)3(C5H10NO2S), C 45.0; H 8.3; N 3.8; S 8.6. IR for Ti(OiC3H7)3(C5H10NO2S), cm-1: 3311 m, 3255 w, 3160 w, 1651 s, 1595 m, 1581 m, 1366 sh, 1313 s, 1271 w, 1257 m, 1162 m, 1126 s, 1114 s, 1031 m, 1009 s, 978 s, 927 s, 847 m, 816 m, 799 w, 623 m, 587 m, 556 m, 492 m, 481 sh, 464 w, 436 w. The chemical identity of the produced titanium precursors was confirmed also by X-ray single crystal studies (see below). Same procedure was used for the preparation of zirconium and aluminum-based precursors, using 1 mL of 70% solution of Zr(OnPr)4 in nPrOH or about 0.5 g of solid Al(OiPr)3 in 5 mL toluene. The concentrated solutions were then evaporated in vacuum, leaving dry yellowish powders that were subsequently redissolved in toluene to achieve desired concentration for further use. Crystallography. The data collection for four isomers of titanium propoxide complexes with penicillaminate-ligand was carried out at room temperature in sealed nitrogen-filled glass capillaries to prevent decomposition in ambient atmosphere. The structures were solved by direct methods, the coordinates of the majority of non-hydrogen atoms were extracted from the primary solutions and those for the missing atoms were found in subsequent Fourier syntheses. All non-hydrogen atoms were refined by full-matrix techniques first in isotropic and then in anisotropic approximations. The coordinates for the hydrogen atoms were calculated geometrically and included in the final solution in isotropic approximation using a riding model. The details of data collection and structure refinement are summarized in Table TS1 (see Supporting Information). Synthesis of Metal Oxide Microparticles. For preparation of the particles, a volume of 1.5 mL of 5 wt vol % solution of a precursor was quickly immersed using a standard 2.5 mL polypropylene syringe into 7.5 mL of Millipore water through a needle with a chosen inner diameter (as controlled later by SEM). The use of 21 gauge needles resulted normally in mostly 30-45 μm particles, 33 gauge needles in mostly 1-2 μm ones, and 28 gauge ones in intermediate 12-15 μm particles, which opened a possibility for size control of the produced material. The synthesis is based on spraying of a hydrocarbon solution from a syringe into water. These are the spray droplets that are transformed into porous spherical particles. No stirring of the reaction mixture has been applied; instead, the mixture was quickly poured out onto a Petri dish and dried at 50 °C in an aerated oven. Langmuir 2010, 26(12), 9809–9817
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Figure 1. Proposed mechanism for the formation of diatom-like microparticles: the process starts with formation of a dense membrane, consisting of coalesced nanoparticles on the surface of the droplets of hydrocarbon solvent. The membrane permits diffusion of the water inside the formed vesicles and the small droplets of water solution covered by new membrane layer emerge on the inside of the outer membrane (to the left). Diffusion of water from outside and the amphiphilic ligands from inside create osmotic pressure leading to coalescence and explosion of the droplets of aqueous solution, which leads to development of an open pore structure (middle). Complete transformation of the metal-organic precursor into porous oxide and subsequent evaporation of the hydrocarbon solvent result in formation of a hierarchically porous microparticle.
Radiological Studies of Model Drug Adsorption and Delivery. The study of the adsorption and release of phosphory-
lated biomolecules, carried out using 32P-marked ATP as model compound, was performed by Ridgeview Instruments AB, exploiting LigandTracer technology.28 A portion of the material was deposited on a PMMA Petri dish as dispersion in toluene and immobilized by drying in air. A reference solution (3 mL, 10% ethanol in water) was added to the dish that was mounted in an inclined position in the instrument and subjected to rotation so that the immobilized material was periodically wet by solution, and its β-emission was registered immediately afterward. 100 μL of 32P-marked ATP solution (resulting in ∼535 pM ATP in the dish) was added after 10 min, and one more portion of the same volume (resulting in ∼1.04 nM ATP in the dish) after 200 min, corresponding to the start of saturation in the observed adsorption (see Supporting Information). When the adsorption equilibrium was achieved (after 500 min), the mother liquor was replaced by the reference solution and the decrease of radioactivity in the material was followed in the same way.
Preparation of Chiral Penicillamine-Coated TiO2 Nanoparticles. 0.058 g of titanium Penicillamine-propoxide precursor was dissolved in 50 mL of isopropanol and heated to reflux under stirring after been bubbled with Ar for 30 min. At 100 °C, a few drops of ethylenediamine were added followed by 1 mL of freshly prepared 2 M NaOH solution. A white precipitate was immediately observed, and the solution was refluxed for 1 h before being allowed to cool. The solution was centrifuged, and the precipitate was redispersed in isopropanol and examined by UV-vis and CD spectroscopy. The CD spectra have been recorded using Jasco J-810 spectropolarimeter in a 1 cm quartz cell. All CD experiments involved 12 scans and were run at 50 nm/min, while subtracting the baseline scan following the standard procedure.
Results and Discussion The synthesis of porous metal oxide structures is both a simpler and a more complicated process at the same time, compared to that of silica. The hydrolysis-polycondensation of metal alkoxides results directly in rapid formation of well-defined micelle nanoparticles23,24 with the size of about 3-5 nm stabilized in solution through the interaction of the ligands, located on their surface, with the solvents.29,30 The particles self-assemble spontaneously on heterogeneous surfaces, forming dense and smooth (28) Bj€orke, H.; Andersson, K. Appl. Radiat. Isot. 2006, 64, 901–905. (29) Kessler, V. G.; Spijksma, G. I.; Seisenbaeva, G. A.; Ha˚kansson, S.; Blank, D. H. A.; Bouwmeester, H. J. M. J. Sol-Gel Sci. Technol. 2006, 40, 163–179. (30) Kessler, V. G. J. Sol-Gel Sci. Technol. 2009, 51, 264–271. (31) Werndrup, P.; Verdenelli, M.; Chassagneux, F.; Parola, S.; Kessler, V. G. J. Mater. Chem. 2004, 14, 344–350.
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coatings, when the ligands are predominantly hydrophobic29,31 or predominantly hydrophilic,32 which explains the external morphology typical for these materials, i.e., dense and smooth surface of the gels.33 The size and the spheroid shape of the constructions to be assembled via hydrolysis-polycondensation can thus easily be controlled through introduction of droplets of a (relatively viscous) concentrated precursor solution in a nonmixing solvent. To obtain open porosity, it was necessary, however, to involve ligands, possessing pronounced amphiphilic properties to enable the diffusion of water into or out from the droplets. For this role, we have chosen amino acids with additional chemical functions, such as, for example, penicillamines. We were especially interested in application of penicillamines for the synthesis of microparticles for drug delivery in view of the established activity of these compounds as medicines, which permitted construction of a drug delivery system in situ. In particular, D-Penicillamine is broadly used as antirheumatic medicine34 and is applied also for treatment of severe cases of neonatal hyperbilirubinemia in infants.35 The exact size control in the synthesis of microparticles was achieved more easily using a solution of a metal alkoxide, modified by an amino acid, in a hydrophobic solvent (with water diffusing into the droplets and away from the alcohols). We used for this purpose volatile hydrocarbons, such as hexanes or pentanes (petroleum ether), exploiting their evaporation on drying as an additional guarantee for opening of the pores on the surface of the produced porous microparticles. The procedure of synthesis was extremely simple and consisted of injection of the precursor solution into pure, distilled water by a syringe with controlled needle opening size. The proposed formation mechanism for the microparticles is illustrated in the Figure 1. When a droplet containing solution of an alkoxide, modified by an amphiphilic amino acid in a nonpolar solvent, is immersed into water, a layer of primary oxide micelles immediately covers its surface. The alcohol, liberated through hydrolysis, and the amino acid are highly water soluble but cannot penetrate the oxide membrane thus formed (see ref 32 for details), which causes enhanced spontaneous diffusion of water into the formed sphere. The aqueous solution instantly forms droplets on the inner side of the sphere. The droplets are in (32) Kessler, V. G.; Seisenbaeva, G. A.; Ha˚kansson, S.; Unell, M. Angew. Chem., Int. Ed. 2008, 47, 8506–8509. (33) Thouvenel-Romans, S.; Steinbock, O. J. Am. Chem. Soc. 2003, 125(14), 4338–4341. (34) Elling, H.; Rasmussen, O. S.; Elling, P. Ugeskr. Laeger 1984, 146, 1924– 1927. (35) Hansen, T. W. R. Expert Opin. Pharmacother. 2003, 4, 1939–1948.
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Figure 2. Microscopic characterization of the produced microparticles: SEM views of whole particles (produced in the same batch) at magnification 1000, 1500, and 2000 and of a typical cross section.
turn immediately covered by the primary oxide particles, which again form a semipermeable membrane. Further diffusion of water from outside creates increased osmotic pressure within the droplets of the aqueous solution, which crack, leaving a stream of the aqueous solution out from it. If the stream goes into the water medium, it leaves an open hole in the outer membrane of the sphere. If it goes inside, a new membrane of aggregated oxide particles then immediately covers it, and the growth of what will then become a macropore continues. The proposed mineralization mechanism appears to be very much analogous to the growth of a “Chemical Garden”, produced by placing crystals of highly soluble transition metal salts into a sodium silicate solution.33 In the present case, however, the osmotic pressure forces the “branches” to grow inside the droplets. The specific effect of an amphiphilic amino acid as modifier lies in its ability to stabilize the forming droplets as surfactant and to prevent densification of the membranes. The eruption of the droplets of aqueous medium into the organic solvent is competing with their coalescence, which results finally in correlation of the size of the growing macropores with that of the radius of the sphere (see below), creating morphology resembling that of a diatom or a radiolarian. The size of the forming microparticles (see Figure 1) is controlled just by the size of the droplets of the organic phase in the range from ∼1 up to ∼50 μm. The sizes of the openings of the pores on the surface correlate thus with the radius of a “diatom”, just as they do in the real biological objects. In the bigger ones of about 45 μm, they are in the range 0.4-0.6 μm; in the 30 μm ones, they are 0.25-0.36 μm, and in about 1 μm ones, they are 0.1-0.15 μm (see Figure 2). The produced objects have a distinctly hierarchical construction. The formation of the solid oxide material occurs through aggregation of the primary nanoparticles, precipitating from solution. The TEM images (Figure 3) show that after mechanical crossing and 9812 DOI: 10.1021/la1000683
subsequent ultrasound treatment the “diatoms” are split into smaller aggregates of dense spherical constructions, consisting in turn of much smaller primary particles, even spherical in shape. The smallest pores observed in the structure via nitrogen adsorption experiments are clearly originating from the voids between the aggregated primary particles (see Supporting Information FS6-FS8). The AFM images of the surface of microparticles show distinctly the “craters” produced by eruptions of the droplets of aqueous medium back into water (Figure 4). The appearance of the “diatoms” is essentially the same, independently, whether they are produced via insertion of precursor solutions into water at room temperature or on boiling. Their chemical composition differs, however, significantly, dependent on the conditions of their synthesis. The particles produced at room temperature are true hybrids, still containing practically all the amino acid ligands inherited from the precursor. The organic ligands can then constitute over 60 wt % of the total sample. The “diatoms” obtained on boiling retain only about 20% of the initial amount of ligands. The estimation of the surface area of the material produced at room temperature, made in approximation of a monolayer formation for the residual amino acid ligands, provides values in the 260-300 m2/g range, correlating well with the literature data for those obtained by mercuroporosimetry for the gels, obtained by hydrolysis of unmodified metal alkoxides in aqueous medium.19 The results of specific surface measurement (BET) and pore volume determination (at p/p0 = 0.995) by nitrogen sorption and the estimation of the pore volume and pore size distribution by thermoporosimetry36 with o-xylene37 correlate with each other (36) Baba, M.; Nedelec, J. M.; Lacoste, J. J. Phys. Chem. B 2003, 107, 12884– 12890. (37) Billamboz, N.; Baba, M.; Grivet, M.; Nedelec, J. M. J. Phys. Chem B 2004, 108(32), 12032–12037.
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Figure 3. TEM view of the powder prepared by mechanical crossing of the microparticles in a mortar and subsequent ultrasonic distribution of the obtained powder in water.
Figure 4. AFM image of the surface of a 45 μm particle and the profile of its typical cross section.
and show that the smallest mesopores are essentially filled in this case by the residual organics and chemically (through hydrogen bonding) adsorbed water. The pore volume for beads with average size of about 1 μm is 0.20 cm3/g with the major mesopore volume fraction constituted by pores with the diameter of about 10 nm (Figure 5a; the unambiguous deconvolution of the observed broad signal could not be achieved). For the beads with average size of about 30 μm, the pore size distribution is more Langmuir 2010, 26(12), 9809–9817
complex with major contribution from the pores of 20, 56, and 326 nm in diameter (Figure 5c). The surface accessible for nitrogen adsorption for these objects is only 28 m2/g. The organic component can be removed through thermal treatment in air at 500 °C with conservation of the typical appearance of the particles (Figure FS1, Supporting Information); only a small fraction collapses or breaks down. The thermal removal of the organic ligands apparently decreases the true surface area but DOI: 10.1021/la1000683
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Figure 5. Thermogram of crystallization of o-xylene filling 1 μm beads before (a) and after (b) calcination and for 30 μm beads before (c) and after (d) calcination. The main peaks have been converted into pore diameters as shown on the graphs. The peak at -27 °C (b,c,d) corresponds to crystallization of free o-xylene remaining outside the pores.
more than doubles the area available for nitrogen sorption to 55-60 m2/g. The total volume of mesopores increases to 0.24 cm3/g and to 0.33 cm3/g for beads with the sizes 1 and 30 μm, respectively. The average size of the available pores increases also with most pronounced pore diameters of 14, 54, and 240 nm for 1 μm beads (Figure 5b) and 35, 120, and 1116 nm for the 30 μm ones (Figure 5d). The thermal treatment in an inert atmosphere is associated with carbonization and is not able to remove the organic content completely. The amino acid ligands are absorbed on the surface of the “diatoms” via chemisorption mechanism, involving most probably the surface chelation. The 1H NMR data for the penicillamine-loaded titania show that the signals from the ligand adsorbed on the material are appreciably broadened and shifted upfield compared to the free penicillamine. It is important to note that because of the stability of these surface complexes the amino acids are not released from the diatoms on storage in pure water. The SEM-EDS study of the material stored under HPLC-grade water (pH = 7.0) or under D2O revealed unchanged morphology together with unchanged sulfur content for penicillamine-derived products. The release of the adsorbed amino acids occurs, however, under modeled body fluid conditions, corresponding to tissues suffering from inflammation. The freshly obtained microparticles immersed into a solution of trisodium citrate and the parent amino acid (with the concentration of about 15 mM in relation to both) lose 90% of the adsorbed amino acid within 2 weeks. The speed of the release increases with the increased concentration of polycarboxylate ligands such as citrate or lactate. The increased concentration of these species is commonly associated with active inflammatory processes in a human body, which demonstrates a possibility to apply the titania microparticles as “smart drug release” sources. The release of the amino acid content in the presence of chelating carboxylate ligands is 9814 DOI: 10.1021/la1000683
associated with clearly observed biodigestion of the material. The residues from microparticles retrieved from a model body fluid medium appear fragmented with macroporosity partly “cured” and replaced by mesoporosity (see Figure 6). The heat-treated “diatoms” display high selectivity in adsorption of molecules from solutions. No observable adsorption of the amino acids occurs, while the affinity to phosphate ligands, typical of titania, remains pronounced and permits binding of the amounts of phosphonic acids equal to that of the amino acids in the initial non-heat-treated material. This opens prospects for application of the diatoms as scaffolds for drug delivery: shortchain amino phosphonic acids such as 2-aminoethyl-phosphonic acid and 3-aminopropyl-phosphonic acid are recognized as agonists of GABA receptors.38 Slow release of these molecules at concentrations of 100 nM (10-7 M) is considered sufficient for stabilization of insulin production in pancreas and can be applied in treatment of diabetes.39 Both compounds are strongly binding to the pre-heat-treated titania microparticles as can be clearly seen from EDS analysis (Figure 6d). The binding occurs via phosphonate moiety and is very distinctly manifested in both 1H and 31P NMR spectra: the signals are broadened and shifted upfield for both proton and phosphorus resonance for the adsorbed ligand compared to the freshly added one (Figure FS2, Supporting Information). A special interest in adsorption and release of biomedicines lies in the possibility of immobilization and slow release of DNA and RNA in biological systems. We have chosen adenosine triphosphonic acid (ATP) in its neutral form (disodium salt) as a model nucleotide for the study of retention and release by titania (38) Qian, H.; Dowling, J. E. J. Neurosci. 1994, 14, 4299–4307. (39) Morgado, C.; Pinto-Ribeiro, F.; Tsavares, I. Neurosci. Lett. 2008, 438, 102– 106.
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Figure 6. SEM view of microbeads stored in solutions: (a) non-heat-treated penicillamine-TiO2 “diatoms” in pure water, 1 week; (b) nonheat-treated beads in 0.015 M sodium citrate þ 0.015 M L-penicillamine, 2 weeks; (c) beads heat-treated at 500 °C for 2 h in 0.050 M L-penicillamine, 1 week; (d) beads heat-treated at 500 °C for 2 h in 0.015 M 2-aminoethylphosphonic acid, 1 week.
microparticles. The data, obtained in the studies of immobilization and release of 32P-marked ATP (see Figure 7), have shown that the affinity of the material to phosphorylated ligands is much higher than to amino acids. The adsorption of phosphorylated molecules from solution is generally following a Langmuir isotherm, showing that the competition from the originally adsorbed ligands is insignificant. The subsequent release into an inert medium after the initial period of two days follows first-order kinetics with the rate constant as low as k = 0.0015 h-1. Combination of a relatively long release time with enhanced release on contact with inflammated tissues makes the produced material highly attractive for application in medical implants. Material with analogous morphology and pronounced ability to selectively absorb biomolecules with phosphate or phosphonate anchors has been obtained even from zirconium and aluminum alkoxides modified with penicillamines (see Figure FS3, Supporting Information). One of the most important advantages in the proposed approach to the synthesis of nanoporous substrates lies in the possibility to provide them with a chiral and stereospecific surface. The surface-shaping ligands applied in the proposed approach are R-amino acids or their oligomers, small proteins, possessing a stereospecific shape. The molecular precursors of oxide materials, metal alkoxides, normally form centrosymmetric molecules.40 Introduction of chiral ligands into the coordination spheres of metal atoms provides, in the majority of cases, the (40) Turova, N. Y.; Turevskaya, E. P.; Kessler, V. G.; Yanovskaya, M. I. The Chemistry of Metal Alkoxides; Kluwer AP: Dordrecht, 2002.
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possibility to obtain chiral derivatives. The alkoxide complexes with chiral heteroligands and, in particular, with R-amino acid residues are extremely scarce. Only two structures of alkoxide R-amino carboxylates, namely, a chiral Fe12(OMe)12(Proline)1212þ(ClO4-)1241 and centrosymmetric Ti2(OEt)6(Glycine)2,42 can be found in the Cambridge Crystal Structure Database. The amino acid derivatives of aluminum and zirconium are unknown. We have recently reported the structure of the first chiral Ti-based representative of this family.32 In the present work, we have systematically investigated the structures of titanium alkoxides monosubstituted with both D- and L-penicillamine and have found that all of them form chiral crystal structures, built up of non-centrosymmetric molecules, where the amino acid residue is attached to the Ti-center as an N,O-chelate (see Figure 8). This feature is transferred to the surface of the formed oxide nanoparticles through the templation effect. It has previously been envisaged in the synthesis of chiral cadmium sulfide nanoparticles as an example that the chirality of the nanoparticle surfaces introduced through the templation by a chiral ligand can be followed by circular dichroism (CD) spectroscopy.43,44 Formation of chiral surface could be demonstrated unequivocally in the present case applying CD spectroscopy to colloidal suspension of titanium dioxide particles (see Figure 9), which have (41) Abu-Nawwas, A. A. H.; Cano, J.; Christian, P.; Mallah, T.; Garajarman, G.; Teat, S. J.; Winpenny, R. E. P.; Yukawa, Y. Chem. Commun. 2004, 314–315. (42) Schubert, U.; Tewinkel, S.; Moller, F. Inorg. Chem. 1995, 34, 995–997. (43) Moloney, M. P.; Gun’ko, Y. K.; Kelly, J. M. Chem. Commun. 2007, 3900– 3902. (44) Elliott, S. D.; Moloney, M. P.; Gun’ko, Y. K. Nano Lett. 2008, 8, 2452– 2457.
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Figure 7. Design of a LigandTracer test setup (to the left) and the accumulation-release activity curve for ∼30 μm beads with 32P-marked ATP as a ligand (to the right). The particles are attached to a polycarbonate Petri dish via deposition of a suspension in toluene with subsequent drying. The inclined dish is rotating and particles are wet periodically by the liquid phase. When the latter is a solution of radioactively marked ligand, the radioactivity of the particles is increasing. When the solution is replaced by a model aqueous phase, the activity is slowly decreasing via release of the ligand.
Figure 9. CD spectrum of L-Pca templated titanium dioxide particles in isopropanol.
(Rhodamine C). This behavior is typical of the non-heat-treated sol-gel derived titania and is due to the presence of an amorphous layer on their surface causing recombination of electron-hole pairs, produced by UV radiation.30,32 Chirality is one of the most important factors of molecular recognition, and therefore, chiral compounds play a very significant role in chemistry, biology, and medicine. Chiral surfaces are very important in a broad range of technological applications such as enantioselective catalysis, nonlinear optical devices, sensors, smart coatings, and chiral separations. In addition, many biological processes involve chiral interactions with surfaces, such as cell adhesion and protein adsorption.45,46 Therefore, the possibility to obtain high-temperature stable supports with chiral surface opens great prospects for production of highly selective stereospecific materials for catalysis and biological separation. High surface area and hierarchical porosity in combination with active and selective sorption properties and a possibility to provide the created surface with chirality makes the produced hierarchically porous microparticles an attractive alternative to mesoporous silicas8 and STARBON47,48 in preparation of catalyst supports. Figure 8. Molecular structures of Pca-substituted titanium alkoxides (A, [Ti(OiPr)3(L-Pca)]2; and B, [Ti(On)Pr)3(L-Pca)]2).
shown a clear CD response. The spectrum of the chiral templated nanoparticles differs considerably from that of the free templating ligand in the same medium (Figure FS4, Supporting Information). The intensity of the signal decreases noticeably in the region 280-400 nm, because of the UV-absorption typical of titania nanoparticles. The particles, however, have not revealed any noticeable activity in photochemical oxidation of model dye 9816 DOI: 10.1021/la1000683
Conclusions Solutions of metal alkoxide precursors, modified with amphiphilic amino acid ligands, in hydrocarbon solvents provide on (45) Raval, R. Chem. Soc. Rev. 2009, 38, 707–721. (46) Mastai, Y. Chem. Soc. Rev. 2009, 38, 772–780. (47) Budarin, V. L.; Clark, J. H.; Luque, R.; Macquarrie, D. J.; Koutinas, A.; Webb, C. Green Chem. 2007, 9, 992–995. (48) Budarin, V. L.; Clark, J. H.; Luque, R.; Macquarrie, D. J. Chem. Commun. 2007, 6, 634–636.
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hydrolysis, when inserted into aqueous medium, hierarchically porous microparticles of metal oxides with high open porosity. The particles contain a considerable fraction of the heteroligands adsorbed on their surface. The ligands are practically not released at all in water, but are readily liberated on biodigestion of titania microparticles occurring in media modeling body fluids in tissues suffering from inflammation and oxidative stress. The produced material reveals high thermal stability and a strong selective trend toward adsorption of ligands containing a phosphate or phosphonate moiety, which makes the obtained microparticles interesting as a potential matrix for smart drug release. Application of chiral ligands offers chiral precursors, which open an approach to the synthesis of oxide materials possessing chiral surface. Acknowledgment. The authors express their gratitude to the Swedish Research Council (Vetenskapsra˚det) for the support of this work. Bruker Nano is gratefully acknowledged for carrying out the AFM characterization of the produced materials.
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Article
Rolf Andersson is gratefully acknowledged for the help with NMR-experiments. Karl Andersson and Ridgeview Instruments AB are gratefully acknowledged for the generous help with in vitro studies, applying radioactive markers. AHD would like to give thanks to the “Conseil Regional de la Martinique” for the financial support. We also thank Science Foundation Ireland RFP scheme and CRANN for financial support. Supporting Information Available: Details of X-ray single crystal experiments (Table TS1), thermal behavior of the microbeads (Figure FS1), NMR characterization of binding of the GABA receptor antagonist - aminoethyl-phosphonic acid to the TiO2 beads (Figure FS2), reference CD spectra of ligands used for preparation of chiral surfaces (Figure FS3), details of radiological experiments (Figures FS4, FS10), details of nitrogen adsorption experiments for different types of TiO2 beads (Figure FS5-FS7), and characterization of ZrO2 and Al2O3 beads (Figures FS8,FS9). This material is available free of charge via the Internet at http://pubs.acs.org.
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