Characterization and Surface Properties of Amino-Acid-Modified

Solvent renewal was carried out once a day for 7 days and the solutions were stirred during dialysis. .... It is also interesting to note that the cry...
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Langmuir 2007, 23, 12233-12242

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Characterization and Surface Properties of Amino-Acid-Modified Carbonate-Containing Hydroxyapatite Particles Kevin S. Jack,* Timothy G. Vizcarra, and Matt Trau Centre for Nanotechnology and Biomaterials, LeVel 5 East, Australian Institute for Bioengineering and Nanotechnology (AIBN), UniVersity of Queensland, St Lucia, QLD 4072, Australia ReceiVed June 22, 2007. In Final Form: September 17, 2007 The surface properties (nature, strength, and stability of interaction of functional groups) and bulk morphologies of a series of amino-acid-functionalized carbonate-containing hydroxyapatite (CHA) particles were investigated. It was found that the amino acids were both occluded in and presented on the surface of the CHA particles. Furthermore, their presence enhanced particle colloidal stability by retardation of Ostwald ripening and in some cases increasing the magnitude of the ζ-potential. Measurements of adsorption isotherms and ζ-potential titrations have shown that the amino-acid-surface interactions are weak and reversible at pH 9 and consistent with a model in which the carboxyl terminus interacts with calcium ions in the CHA lattice. Complexities in adsorption behavior are discussed in terms of different adsorption mechanisms that may be prevalent at different pHs.

Introduction Hydroxyapatite (HA), [Ca10(PO4)6(OH)2], is one of the most stable forms of the calcium orthophosphates and the major inorganic component of bone and teeth in mammals.1,2 In nature, stoichiometric HA is very rarely found and it is more often formed with partial substitution by carbonate, Mg2+, Na+, etc. The mechanical and biological properties of pure and substituted HA have been well studied and are the subjects of many publications, including reviews and texts.1-4 Of these properties its relatively high stiffness and yield strength, osteoconductive nature, and biocompatibility (with calcified tissues) has led to much interest into the applications of HA and substituted HAs in the fields of bone tissue engineering and orthopaedic therapies.2,5-9 In addition, the surface adsorption properties of these materials has led to applications in affinity chromatography,10-12 wastewater remediation,13,14 and drug delivery systems.9,15 There exist numerous synthetic strategies for producing HA and substituted HAs including wet precipitation, hydrothermal methods, sol-gel, and solid-state synthesis.1,3,16,17 Recently, Gonzalez-McQuire et al.17 utilized a hydrothermal method in which stable dispersions of ultrafine, rodlike, or prolate ellipsoidal (1) Dorozhkin, S. V. J. Mater. Sci. 2007, 42, 1061-1095. (2) Biomaterials Science: an Introduction to Materials in Medicine, 2nd ed.; Ratner, B. D., Ed.; Elsevier Academic Press: Amsterdam, 2004. (3) Hyroxyapatite and Related Material; Brown, P. W., Constantz, B., Eds.; CRC Press: Ann Arbor 1994. (4) Yaszemski, M. J.; Payne, R. G.; Hayes, W. C.; Langer, R.; Mikos, A. G. Biomaterials 1996, 17, 175-185. (5) Ben-Nissan, B.; Milev, A.; Vago, R. Biomaterials 2004, 25, 4971-4975. (6) Gonzalez-McQuire, R.; Green, D.; Walsh, D.; Hall, S.; Chane-Ching, J.Y.; Oreffo, R. O. C.; Mann, S. Biomaterials 2005, 26, 6652-6656. (7) Ma, P. X. Mater. Today 2004, 30-40. (8) Wei, G. B.; Ma, P. X. Biomaterials 2004, 25, 4749-4757. (9) Choi, A. H.; Ben-Nissan, B. Nanomedicine (London, United Kingdom) 2007, 2, 51-61. (10) Kawasaki, T. J. Chromatogr. 1991, 544, 147-184. (11) Bernardi, G.; Giro, M. G.; Gaillard, C. Biochim. Biophys. Acta 1972, 278, 409-420. (12) Gorbunoff, M. J.; Timasheff, S. N. Anal. Biochem. 1984, 136, 440-445. (13) Barba, F.; Callejas, P. J. Mater. Sci. 2006, 41, 5227-5230. (14) de-Bashan, L. E.; Bashan, Y. Water Res. 2004, 38, 4222-4246. (15) Perkin, K. K.; Turner, J. L.; Wooley, K. L.; Mann, S. Nano Lett. 2005, 5, 1457-1461. (16) Milev, A. S.; Kannangara, G. S. K.; Wilson, M. A. Langmuir 2004, 20, 1888-1894. (17) Gonzalez-McQuire, R.; Chane-Ching, J. Y.; Vignaud, E.; Lebugle, A.; Mann, S. J. Mater. Chem. 2004, 14, 2277-2281.

Scheme 1: Scheme of Amino Acids

(cigar shaped) HA particles (ca. 4-15 nm wide by 30-150 nm long) were prepared in aqueous solution. The synthetic scheme reported employed addition of an aqueous ammonium phosphate solution to an aqueous solution containing calcium nitrate plus an amino acid at pH 9; glycine, alanine, valine, asparagine, serine, lysine, arginine, and aspartic acid were all reported. The particles produced were found to be smaller and of a higher aspect ratio than those of the control sample (without amino acid present) and generally more stable to aggregation. The presence of amino acids at the particle surfaces were confirmed by both FTIR and zeta (ζ) potential measurements. Furthermore, the authors showed that it was possible to cross couple the amino-acid functionality at the surface of the particles to produce aggregated clusters with some degree of preferential elongation along the long axes of the particles. In this work we report a detailed investigation of the nature and stability of the interaction of a series of amino acids with the surface of carbonate-containing hydroxyapatite (CHA) particles using a range of techniques including measurements of the adsorption isotherms of the amino acids, ζ-potential titrations, surface characterization by XPS, and bulk characterization by XRD and TGA. The amino-acid-functionalized CHA particles were prepared by the method of Gonzalez-McQuire et al.,17 and further to their initial findings, it is demonstrated that the amino acids are both adsorbed at the CHA surface and occluded within the primary particles. Moreover, measurements of adsorption

10.1021/la701848c CCC: $37.00 © 2007 American Chemical Society Published on Web 10/27/2007

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isotherms and ζ-potential titrations have shown that the interaction between amino acids and the surface of CHA is weak and reversible and that the stability and functionality provided by the amino acids can be readily lost during purification procedures. The mechanism of stabilization of the CHA particles was also investigated in further detail, and it is shown that in addition to providing increased charge stabilization in the case of certain amino acids, retardation of the rate of Ostwald ripening (and hence a reduction in interparticle attractive forces and the rate of sedimentation) also plays a significant role. This final point is surprisingly not well stated in the literature, although it is well recognized as the basis of methods to control the shape and morphology of HA and CHA particles; see, e.g., refs 18-21. Measurements of adsorption isotherms of various amino acids,22-28 small organic molecules,29-33 as well as macromolecules (peptides, proteins, lysosomes, etc.)25,34-39 onto the surface of HA, CHA, or fluorinated HA particles have been previously reported. Additionally, the presence of different amino acids in solution has been shown to significantly reduce the kinetics of crystallization of HA from a supersaturated solution onto seed particles.27,28,40-44 In general, the authors apply the Langmuir model to the experimental isotherms or particle growth data to quantify the relative affinities and capacities of the adsorbents onto HA surfaces. This can be used to better understand the efficiency of HA columns for separating peptides and related compounds or provide insight into the functional groups that may be interacting with the HA surface. Alternatively the adsorption of serine onto HA at pH 7 has been modeled with a Freundlich isotherm; it was suggested that this was indicative of the weak nature of the interaction between the amino acid and the surface.24 Variations in the nature of the particles used (e.g., the degree of substitution by carbonate or other ions) or conditions at which the isotherms were measured (e.g., pH) make it difficult, however, (18) Sonoda, K.; Furuzono, T.; Walsh, D.; Sato, K.; Tanaka, J. Solid State Ionics 2002, 151, 321-327. (19) Oener, M.; Dogan, O. Prog. Cryst. Growth Charact. Mater. 2006, 50, 39-51. (20) Zhang, H. G.; Zhu, Q. Chem. Lett. 2005, 34, 788-789. (21) Zhang, H. G.; Zhu, Q.; Wang, Y. Chem. Mater. 2005, 17, 5824-5830. (22) Aoba, T.; Moreno, E. C. J. Colloid Interface Sci. 1985, 106, 110-121. (23) Kresak, M.; Moreno, E. C.; Zahradnik, R. T.; Hay, D. I. J. Colloid Interface Sci. 1977, 59, 283-292. (24) Benaziz, L.; Barroug, A.; Legrouri, A.; Rey, C.; Lebugle, A. J. Colloid Interface Sci. 2001, 238, 48-53. (25) Garcia-Ramos, J. V.; Carmona, P.; Hidalgo, A. J. Colloid Interface Sci. 1981, 83, 479-484. (26) Misra, D. N. J. Colloid Interface Sci. 1997, 194, 249-255. (27) Moreno, E. C.; Kresak, M.; Hay, D. I. Calcif. Tissue Int. 1984, 36, 48-59. (28) Spanos, N.; Klepetsanis, P. G.; Koutsoukos, P. G. J. Colloid Interface Sci. 2001, 236, 260-265. (29) Misra, D. N. J. Biomed. Mater. Res. 1999, 48, 848-855. (30) Chirdon, W. M.; O’Brien, W. J.; Robertson, R. E. J. Biomed. Mater. Res., Part B 2003, 66B, 532-538. (31) Mangood, A.; Malkaj, P.; Dalas, E. J. Cryst. Growth 2006, 290, 565570. (32) Vega, E. D.; Narda, G. E.; Ferretti, F. H. J. Colloid Interface Sci. 2003, 268, 37-42. (33) Misra, D. N. Langmuir 1988, 4, 953-958. (34) Misra, D. N. J. Colloid Interface Sci. 1996, 181, 289-296. (35) Barroug, A.; Lemaitre, J.; Rouxhet, P. G. Colloids Surf. 1989, 37, 339355. (36) Barroug, A.; Fastrez, J.; Lemaitre, J.; Rouxhet, P. J. Colloid Interface Sci. 1997, 189, 37-42. (37) Barroug, A.; Lernoux, E.; Lemaitre, J.; Rouxhet, P. G. J. Colloid Interface Sci. 1998, 208, 147-152. (38) Tsortos, A.; Nancollas, G. H. J. Colloid Interface Sci. 2002, 250, 159167. (39) Capriotti, L. A.; Beebe, T. P., Jr.; Schneider, J. P. J. Am. Chem. Soc. 2007, 129, 5281-5287. (40) Koutsopoulos, S.; Dalas, E. J. Colloid Interface Sci. 2000, 231, 207-212. (41) Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 2000, 217, 410-415. (42) Koutsopoulos, S.; Dalas, E. Langmuir 2000, 16, 6739-6744. (43) Koutsopoulos, S.; Dalas, E. J. Cryst. Growth 2000, 216, 443-449. (44) Koutsopoulos, S.; Dalas, E. Langmuir 2001, 17, 1074-1079.

Jack et al. Table 1. pKa Values and Isoelectric Points (pI) of the Amino Acids Used in This Work; from Nelson and Cox45 amino acid glycine alanine serine aspartic acid lysine

sample code GLY ALA SER ASP LYS

pKa COOH

pKa NH2

2.34 2.34 2.21 1.88 2.18

9.60 9.69 9.15 9.60 8.95

pKa side chain

pI

3.65 10.53

5.97 6.01 5.68 2.77 9.74

to compare the affinities or maximum adsorbed amounts obtained from the various authors and hence for a wide range of aminoacid structures. Moreover, while Koutsopoulos and Dalas40-44 studied the effects of a wide range of amino acids on the rate of crystallization of HA, it is not clear if the parameters obtained from Langmuir-type fits to these data are directly comparable to those obtained from measurements of the adsorption isotherms.28 In addition Misra29 investigated the adsorption of a range of molecules from both aqueous and nonaqueous solution and also studied the reversibility or irreversibility of these adsorption processes. It was concluded that adsorption of low molecular weight solutes from an aqueous solution generally involves an ion-exchange process and that additional processes such as precipitation of salts of the solute can lead to the apparently anomalous adsorption parameters. The amino acids studied in this work are shown in Scheme 1, and some of the physical properties45 of these amino acids are summarized in Table 1. The isoelectric point of the amino acids (pI) shown in this table represents the pH at which the total number of positive and negative charges on the amino acid are balanced; i.e., at pH > pI there will be a net negative charge. The amino acids were chosen to represent a series of simple structures with increasing size (GLY < ALA < SER) and sidegroup charge (ASP negative, GLY neutral, LYS positive). Experimental Section Materials. Carbonate-Containing Hydroxyapatite (CHA). The syntheses of CHA and amino-acid-functionalized CHA particles were performed in a manner similar to that described by GonzalesMcQuire et al.17 Typically, 35.29 g of Ca(NO3)2·4H2O (Ridedel-de Hae¨n, AR grade) and 2 mol equiv of the respective amino acid (Sigma, AR grade) relative to the amount of Ca2+ was dissolved in 150 mL of milliQ water. The pH was adjusted to 9 via addition of 28% NH3(aq) solution (Ajax, AR grade), and the mixture was stirred and heated to 80 °C in an oil bath. A 3.37 mL aliquot of 85% ortho-phosphoric acid (Fluka, AR grade) was dissolved in 150 mL of milliQ water, and the pH was adjusted to 9 with 28% NH3(aq). This was added instantaneously to the Ca2+/amino-acid mixture, after which a white solid was observed to precipitate immediately from the solution. The particles were aged by stirring and refluxing at 80 °C for 18 h. With the exception of CHA-ASP, this produced CHA dispersions that did not sediment for several weeks. CHA Washing Procedure. CHA particles were purified by centrifugation at 2000 rcf for 1 min, removal of the supernatant, and resuspension in fresh solvent of interest. Typical washing procedures were repeated 5 times to ensure removal of excess amino acids. Particle Dialysis. A 10 mL aliquot of freshly synthesized CHAGLY particles was purified by dialyzing against excess glycine solution, prepared by dissolving 7 g of glycine into 1 L of distilled water and adjusting the pH to 9 by the addition of 0.25 M KOH. Solvent renewal was carried out once a day for 7 days and the solutions were stirred during dialysis. Measurement of Amino-Acid Adsorption Isotherms. CHA for these experiments was synthesized as above without amino acid present. Particles were collected by centrifugation and washed using the (45) Nelson, D. L.; Cox, M. M. Lehninger principles of biochemistry, 3rd ed.; Worth Publishers: New York, 2000.

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procedure described previously, followed by drying in an oven. The aggregated crystals were then pulverized using a mortar and pestle. The CHA powder was heated in an oven at 500 °C for 24 h to remove residual organic matter. BET analysis revealed the specific surface area of the resultant particles to be 34.5 m2 g-1. Isotherms of the adsorption of the amino acids onto CHA were constructed using a depletion method. The concentration of amino acid in solution before and after equilibration with CHA particles was measured by reaction with a dye and subsequent detection by spectrophotometry (see details below). The difference between these concentrations was taken as the amount adsorbed onto the CHA surface. For each of the amino acids 5 mL of the respective amino-acid solution (concentrations ranging between 1 and 40 mM) was added to 90 mg of particles, and the pH was adjusted to 9 via addition of 0.25 M KOH. The suspensions were then agitated for 24 h, a time determined experimentally to be sufficient for equilibrium to be attained. The suspensions were centrifuged at 4000 rcf for 10 min, after which 5 µL of supernatant was extracted and lyophilized at 0.25 Torr for 4 h to remove water. The concentration of amino acid remaining in the extracted supernatant was determined via reaction with ninhydrin and subsequent measurement of the absorption at 570 nm.46 A 50 µL amount of 80% phenol in ethanol (Fluka), 50 µL of KCN in water/ pyridine (Fluka), and 50 µL of 6% ninhydrin in ethanol (Fluka) were added to each of the lyophilized amino acids, which were then heated at 95 °C for 5 min. A 3.6 mL amount of 60% v/v ethanol (Ajax, AR grade) in water was added to each solution, which was then agitated to ensure complete mixing. The concentration of each solution was determined by measuring the absorbance at 570 nm and using the Beer-Lambert law. Calibration plots were constructed to determine the molar extinction coefficients for the Ruheumann’s Blue complexes and performed for each amino acid to account for the different sensitivities of each amino acid to the ninhydrin reaction. Two replicates of each isotherm were measured and subsequently averaged. Blank experiments indicated that no background response resulted from reaction of ninhydrin with CHA or from light scattering from residual CHA particles. Instrumental Methods. X-ray Diffraction. Samples were analyzed in a Bruker D8 Advanced X-ray diffractometer equipped with a graphite monochromator, copper target, and scintillation counter (detector). Measurements were conducted from 2θ ) 20° to 70° in 0.050° steps using a step time of 12 s per step. The powder patterns were compared with database fingerprints (Powder Diffraction File, second Release 2003) using the DIFFRACplus Evaluation Package. InductiVely Coupled Plasma-Optical Emission Spectroscopy. Particle bulk compositions were determined using a Varian VistaPro CCD Simultaneous ICP-OES spectrometer fitted with a concentric nebulizer and a forward power setting of 1200 W. Samples were dissolved in 5% nitric acid prior to analysis. ζ-Potential Titrations. Electrokinetic measurements were conducted on a Malvern Instruments Nano Series ZS Zetasizer (model ZEN3600) fitted with a 633 nm laser and a MPT-2 autotitration unit. Samples were diluted by a factor of 50:1 in Millipore water with a background electrolyte concentration of 1 mM KCl so that a count rate of 1800-2000 kilocounts per second was maintained. Thirty runs were performed and averaged for each measurement. ζ-potentials were calculated using the Henry equation UE )

2zf(ka) 3η

(1)

where UE is the particle electrophoretic mobility,  is the solvent dielectric constant, η is the solvent viscosity, f(ka) is Henry’s function, and z is the ζ-potential. Smoluchowski’s approximation was used, and f(ka) was taken to be 1.5. Changes in pH were obtained via addition of 0.25 M HCl and 0.25 M KOH. CHA particles were also titrated with 0.25 M K3PO4 as well as 0.25 M CaCl2 in order to understand the effects of these potential-determining ions. (46) Friedman, M. J. Agric. Food Chem. 2004, 52, 385-406.

Infrared Spectroscopy. IR spectra were recorded from 4000 to 525 cm-1 on a Nicolet Nexus 8700 FTIR spectrometer with a singlereflection diamond Smart Omni-Sampler ATR sampling accessory. Sixty-four scans were recorded at a resolution of 8.00 cm-1 and a mirror velocity of 0.6329 cm-1. BET Analysis. Particle specific surface areas were determined using a NOVA 1200 Quantachrome apparatus. N2 partial pressures were varied between 4.5 × 10-2 to 2.5 × 10-1, and a cross-sectional area of 16.2 Å2 was assumed for nitrogen. Scanning Electron Microscopy. Particle suspensions at concentrations of approximately 0.01% w/w were dropped onto aluminum stubs coated with a carbon adhesive and covered with glass slips. Platinum coating was performed using an EIKO IB-5 Sputter Coater for 3 min, resulting in coat thicknesses of approximately 10 nm. Images were obtained using a JEOL JSM 6300 at an aperture of 4 and an accelerating voltage of 6 kV. UV Measurements. UV absorption measurements were conducted using a Perkin-Elmer Lamda 2 UV/vis spectrometer. Scans were conducted from 800 to 190 nm in data intervals of 1.0 nm and a speed of 1 nm s-1. TGA Measurements. TGA measurements were conducted using a Shimadzu Thermogravimetric Analyzer. Approximately 25 mg of sample was used in each measurement, which was conducted in a nitrogen atmosphere (80 mL min-1). Samples were heated to 600 °C at a rate of 5 °C min-1.

Results and Discussion Particle Synthesis, Characterization, and Morphology. All of the amino-acid systems studied produced visibly cloudy suspensions during the reaction and subsequent aging and stirring process. Suspensions prepared without any amino acid present were observed to form dense white sediments within a few minutes of being stood on the bench after 18 h of maturation. The suspension of CHA-ASP was also observed to sediment when left to stand for a period of hours. The remaining CHA-aminoacid dispersions, however, did not sediment over a period of greater than 2 weeks. Moreover, SEM measurements of the CHA particles precipitated without amino acids showed that the particles were initially of the order of 150 nm in length after 2 h of maturation and proceeded to grow to ∼1-2 µm over a period of 4 weeks. In contrast to the untreated CHA particles, the particles prepared with amino acids were found to not increase significantly in size after the initial maturation process of a few hours. SEM images of the particles produced by the precipitation and maturation (18 h) process are shown in Figure 1. These images were collected from a drop of the sample dispersion which has been rapidly evaporated onto a carbon stub. Aggregation of the particles observed in the images is likely, in part at least, to be a result of the drying process. The approximate mean sizes (length and maximum width) of the particles were determined by averaging ca. 100 primary particles in each of the images and are reported in Table 2. The ratios of calcium to phosphorus (Ca:P) in the particles were measured by both ICPOES and XPS. The ICP method measures the bulk Ca:P, while XPS probes only the top ca. 1-5 nm of the surface of the crystals. For both sets of measurements the particles were washed to remove excess ions and amino acid. These ratios are shown in Table 3. The bulk Ca:P ratio of 1.88 determined for the CHA control sample is larger than the stoichiometric value of 1.67 for pure HA but is in a range which is reported for carbonatesubstituted HA.47 Furthermore, it can be seen that the bulk Ca:P ratio shows little change upon addition of the amino acids. Powder XRD measurements for the particles are shown in Figure 2. These XRD patterns are all consistent with poorly (47) Milev, A. S.; Kannangara, G. S. K.; Wilson, M. A. J. Phys. Chem. B 2004, 108, 13015-13021.

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Figure 1. SEM images of CHA particles prepared in the presence of (a) no amino acid (CHA control sample), (b) 1 M GLY, (c) 1 M ALA, (d) 1 M SER, (e) 1 M LYS, and (f) 1 M ASP. Table 2. Sizes of CHA-AA Particles Determined by SEM sample code

lengtha (nm)

widtha (nm)

aspect ratio (L/W)

none GLY ALA SER ASP LYS

258 ( 6 162 ( 1 96 ( 2 118 ( 3 57 ( 1 123 ( 2

28 ( 1 29 ( 1 30 ( 1 29 ( 1 20 ( 1 32 ( 1

9.2 5.6 3.2 4.1 2.9 3.8

a Mean sizes determined by averaging ca. 100 randomly selected primary particles.

Table 3. Ca:P Ratio and Organic Content of Washed CHA-AA Particles amino acid

Ca:P ICP-OES ( 0.05

Ca:P XPS ( 0.1

organica content (%) ( 3%

none GLY ALA SER ASP LYS

1.88 1.72 1.75 1.85 1.88 1.88

1.6 2.1 1.8 2.0 2.0 1.9

3 10 6 9 20 9

a Samples washed to remove surface- bound molecules, and the organic content is defined as the % mass loss from 100 to 600 °C (see text).

crystalline CHA.17,47 Moreover, FTIR measurements (see Supporting Information) of these particles were performed and found to be consistent with HA containing CO3 substitutions.47-49 The carbonate substitution in these particles is estimated to be ca. 4 wt % from the FTIR spectra and the empirical relationship of Featherstone et al.49 and typical for apatite particles prepared at ambient CO2 concentrations. It is not possible to assess the carbonate content of the HA samples reported by Gonzalez(48) Kumar, R.; Prakash, K. H.; Cheang, P.; Khor, K. A. Langmuir 2004, 20, 5196-5200. (49) Featherstone, J. D. B.; Pearson, S.; LeGeros, R. Z. Caries Res. 1984, 18, 63-66.

McQuire et al.;17 however, it seems likely that the level will be similar given the similarity in the method of preparation. It can be seen from Figure 2 that the peaks were broadened compared to those in the XRD of the CHA control sample, indicating that crystalline domains within the particles functionalized with amino acids are smaller or significantly more disordered than those in the CHA control. Incorporation of the amino acids also increased the fraction of amorphous material in the particles, which can be seen as a broad featureless peak (‘amorphous halo’) under the crystalline reflections in Figure 2. Qualitatively, it can be seen from the broadening of the 002 reflection at 25.9° and the amorphous halo in Figure 2 that the more negatively charged amino acids cause the most retardation or disorder along the long axis, i.e., ASP < SER < GLY ≈ ALA < LYS. The above results show that the additions of the amino acids to the reaction solution affected the particle size, morphology, composition, and stability of the dispersions. Such a result is in line with previous reports in the literature.17 In contrast to the work of Gonzalez-McQuire et al.,17 however, the particles prepared in this work did not show such well-defined needlelike shapes and were found to be larger and of lower aspect ratio (see Figure 1). These differences are presumably due to subtle differences in the methods of preparation. Adsorption of the amino acids onto or into the CHA particles is further confirmed from the appearance of amino-acid-specific bands in the ∼15001700 cm-1 region of the FTIR spectra (see Supporting Information), which correspond to asymmetric stretching and bending of COO- and NH3+ groups.50-54 The high degree of overlap of amino-acid peaks in the 1500-1700 cm-1 region with those of CHA limits the ability to observe shifts in vibrational energies that may occur upon interaction of the functional groups of the amino acids with CHA and its constituent ions. In the case of the CHA prepared in the presence of ASP, a loss of the carboxylicacid stretching vibration (1685 cm-1) is observed. This observation implies that the protonated ‘free’ acid side chains present in ASP are being converted to the carboxylate form and presumably chelating with Ca2+ ions. It is not possible, however, to determine if the interaction of the carboxyl group occurs with calcium that is contained with the CHA crystal or to free calcium ions in solution. The presence of amino acids during precipitation of the particles was found to significantly reduce the dimensions of the CHA particles along their long axis, although they were observed to produce a negligible reduction in the shorter axis with the exception of ASP (see Table 2). In general, the aspect ratio, defined as the length divided by the width, was found to decrease when the amino acids were present in the reaction mixture with only minor variations observed between the different amino acids used. This finding is in contrast to that reported previously; however, the control sample (CHA matured for 18 h) studied in this work was found to be significantly longer (260 nm, cf. 80 nm) and of greater aspect ratio (9.3, cf. 4) than that reported in the work of Gonzalez-McQuire et al.17. It is also interesting to note that the crystalline domain sizes determined by Scherrer analysis in the work of Gonzalez-McQuire et al. are similar in terms of overall sizes and trends with addition of amino acids (50) Lopez Navarrete, J. T.; Hernandez, V.; Ramirez, F. J. Biopolymers 1994, 34, 1065-1077. (51) Rosado, M. T.; Duarte, M. L. T. S.; Fausto, R. Vib. Spectrosc. 1998, 16, 35-54. (52) Vijayan, N.; Rajasekaran, S.; Bhagavannarayana, G.; Babu, R. R.; Gopalakrishnan, R.; Palanichamy, M.; Ramasamy, P. Cryst. Growth Des. 2006, 6, 2441-2445. (53) Selemenev, V. F.; Zagorodni, A. A. React. Funct. Polym. 1999, 39, 5362. (54) Jarmelo, S.; Reva, I.; Carey, P. R.; Fausto, R. Vib. Spectrosc. 2007, 43, 395-404.

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Figure 2. XRD spectra of CHA particles prepared in the presence of 1 M (a) GLY, (b) ALA, (c) SER, (d) LYS, (e) ASP and (f) no amino acid (CHA control sample).

that would be determined if the same analysis were applied to this work. It is possible that the larger overall primary particle sizes measured by SEM in this work are a result of a greater degree of agglomeration of these crystalline domains during the reaction and maturation To determine whether amino acids may be occluded within these particles, the organic contents of the particles were determined by thermogravimetric analysis (TGA). The particles were washed prior to the TGA measurements to remove surfacebound amino acid. The residual organic content is, therefore, assigned to material occluded within the particles. Representative mass-loss curves are shown in Figure 3, and the percent organic material determined from these curves is presented in Table 3. The percent organic material is defined in this work as the difference in mass between 100 and 600 °C divided by the mass at 100 °C to allow for an initial mass loss due to adsorbed water. Moreover, the relatively high uncertainty quoted for the percent organic content is estimated to account for the loss of volatiles observed in the CHA control sample, which are not due to amino acid. It can be seen from this table that a significant amount of organic material (up to approximately 10% of the mass of the particle or 20% in the case of the sample containing ASP) may be trapped within the primary particles. To put these weight fractions into perspective, from the measurement of the adsorption

Figure 3. Representative mass-loss curves measured by TGA for CHA (top-most curve) and CHA initially prepared in the presence of 1 M ALA, SER, and ASP (in descending order from the CHA curve). All of the samples were washed, as described in the text, to remove surface-bound amino acid before the TGA measurements were carried out.

isotherms presented below (see Table 4) it can be calculated that the weight percent of amino acid that could be attributed to

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Figure 4. ζ-potential titrations of CHA (×) and CHA prepared in the presence of 1 M GLY (2), SER (b), ASP (9), ALA (+), and LYS ([). Lines are shown as guides for the eye with CHA shown as a solid line. Samples were measured as fresh reaction sols diluted by a factor of 50 in milliQ water. Table 4. Parameters Obtained from Langmuir Isotherm Fits of Amino-Acid Adsorption onto Plain CHAa amino acid

K (L mol-1)

GLY ALA SER ASP LYS GLY (pH 7) GLY (pH 11)

110 40 31 32 140 36 no adsorption

a

Γmax (×10-6 mol m-2) 5.1 9.4 10.1 6.9 4.6 12.0 no adsorption

Isotherms were measured at pH 9 unless otherwise stated.

surface adsorption to the primary particle is of the order of 1-5.5%; the maximum value is calculated for the SER sample which has the highest adsorbed amount. As noted above, the XRD patterns (Figure 2) for the CHA-amino-acid particles show both an increase in the content of amorphous or noncrystalline material and a significant broadening of the peaks compared with the CHA control sample. Sarig55 hypothesized on the basis of crystallographic data and geometric arguments that ASP could be able to cocrystallize into carbonate-substituted HA. Moreover, occlusion of the amino acid into the CHA particles may also be the origin of the increased amorphous content that can be observed in Figure 2 upon addition of amino acid (particularly in the ASP and SER samples). Particle Stability. In order to better understand the stability of the CHA dispersions in the presence of amino acid, ζ-potentials of the CHA particles were measured as a function of the pH of the as-prepared particle dispersions. These are presented in Figure 4. As noted above, it was observed that the mean size of the particles prepared in the presence of the amino acid remained constant when stored, whereas the control sample was found to increase in size with time due to Ostwald ripening. Additionally, the CHA particles functionalized with GLY, ALA, SER, and LYS were all found to be stable (i.e., they did not sediment) over a period of weeks at pH ≈ 9, while the control sample and the particles prepared with ASP were observed to sediment. From Figure 4 it can be seen that the ζ-potentials at pH 9 are CHA ≈ -10 mV, CHA-ASP ≈ 0 mV, CHA-ALA ≈ CHA-SER ≈ +10 mV, CHA-GLY ≈ +21 mV, and CHA-LYS ≈ +27 mV. The reason for the instability of the CHA-ASP particles at this pH can be seen to be due to the almost zero surface (55) Sarig, S. Bone 2004, 35, 108-113.

Figure 5. ζ-potential titrations of CHA (×) and CHA initially prepared in the presence of 1 M GLY (2), SER (b), ASP (9), ALA (+), and LYS ([) and then dialyzed against milliQ water for 7 days. Lines are shown as guides for the eye. CHA is shown as a solid line.

potential which provides little charge repulsion and hence a low energy barrier to aggregation. This has also been noted by previous authors.17 The magnitude of the surface potential on the CHA control, however, is similar to that of the ALA- and SER-prepared samples, although the CHA sample was observed to readily sediment while the ALA and SER samples were stable over time. This difference in stability is, therefore, due to the overall reduction and stabilization of particle size that is achieved by the presence of amino acid, i.e., the retardation of the Ostwald ripening process that is observed in the CHA sample. Moreover, incorporation of amino acid into the particles would lead to a reduction in the bulk density of the particles. Therefore, such an increase in the observed stability is to be expected as the attractive forces between the particles become weaker with decreasing particle size and density, thereby increasing the overall barrier for particle aggregation.56 In addition, the rate of sedimentation will also decrease as particle size and density decreases. Any additional increase in the magnitude of the surface potential from adsorption of the amino acids would further increase the particle stability. Presumably the reduction of crystal growth is also critical in the reported stabilization of nanometer-sized CHA particles by addition of polyelectrolyte and surfactants. Figure 5 shows the ζ-potential measurements for the particle dispersions following dialysis against Millipore water for 7 days. In addition, it was observed that the samples contained a large amount of sediment following dialysis. It can be seen from this figure that ζ-potentials as a function of pH for all of the samples show similar trends and have a common isoelectric point (iep). Moreover, the magnitudes of the ζ-potentials for all of the samples originally prepared with amino acid are reduced toward that of the pure dialyzed CHA sample. These results suggest that the surface-bound amino acids are in equilibrium with the surrounding solution and are removed to a large extent by the dialysis process. Such a finding is important as the ability to covalently react other moieties to the amino-acid-functionalized CHA surface may be limited by the necessity to remove residual ions and amino acids following the initial preparation stage. Moreover, redispersing of the functionalized particles even after reactive coupling may ultimately lead to a loss of the functionalized layer. The behavior of the ζ-potential titrations seen in Figure 5, i.e., a common iep with differing ζ-potentials, is consistent with (56) Hunter, R. J. Foundations of colloid science; Oxford University Press: Oxford, 2001.

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Figure 6. ζ-potential titration of CHA initially prepared in the presence of 1 M GLY and then dialyzed against 0.1 M GLY solution for 7 days. The line is shown as a guide for the eye.

Figure 8. Adsorption isotherms of (a) ALA ([), GLY (+), and ASP (9) and (b) SER (b) and LYS (2) adsorbing onto CHA (prepared without amino acid) at pH 9. The solid lines show the results of fitting the data to eq 3.

Figure 7. ζ-potential salt titrations of plain CHA with the potentialdetermining ions (a) Ca2+ and (b) PO43- at pH ) 7. Lines are shown as guides for the eye.

dispersions containing particles with a common surface (in this case CHA) but differing concentrations of indifferent ions. For CHA, Ca2+, PO43-, and OH- are known to be potential determining,57,58 and the effect of adding Ca2+ and PO43- to dispersions of CHA at pH ) 7 is shown in Figure 7, while the effect of adding OH- can be observed in Figure 5 (CHA control sample). Note also that the decrease in ζ-potential observed in the CHA upon dialysis (from -10 to -15 mV) is consistent with removal of excess Ca2+ ions from this solution during dialysis. Given that the samples were first purified by dialysis and prepared with a fixed concentration of background electrolyte, it is not (57) Bell, L. C.; Posner, A. M.; Quirk, J. P. J. Colloid Interface Sci. 1973, 42, 250-261. (58) Garcia Rodenas, L.; Palacios, J. M.; Apella, M. C.; Morando, P. J.; Blesa, M. A. J. Colloid Interface Sci. 2005, 290, 145-154.

clear what the exact source of the indifferent ions is. By comparison of Figures 4 and 5, it is possible that the small increase in ζ-potential with increasing pH observed at ca. pH ) 8.5 in the case of the LYS and ALA samples in Figure 5 is due to residual amino acid that has not been completely removed from the CHA by dialysis for 7 days. Given the above finding that significant amounts of the amino acids are likely to be occluded into the crystal and, therefore, only removed by dissolution and reprecipitation of the CHA particles, it may take a significant amount of time to completely remove all of the amino acids. We have not attempted to study the rate of dissolution or the rate of CHA crystal growth in these dialyzed samples. Finally it is noted that removal of ions from the reaction mixture without removal of the amino-acid functional groups and the particle stability can be achieved by dialysis of the reaction mixture against a solution containing an excess of amino acid at pH ) 9. ζ-potential measurements as a function of pH for a solution of CHA particles prepared in the presence of GLY and dialyzed against a 0.1 M solution of glycine at pH ) 9 for 7 days are shown in Figure 6. The approximate trend of the ζ-potential is similar to that of the GLY sample measured in the reaction solution shown in Figure 4, although the magnitudes of the ζ-potentials are reduced compared to those of the nondialyzed sample. This is most likely due to the reduction of the excess ions in the original reaction mixture. Nature of the Adsorption of Amino Acids. In addition to measurements of ζ-potential, adsorption isotherms of the amino acids onto CHA particles which had been prepared without amino acid present were measured to gain insight into the nature of the interaction of the amino acids with the surface of CHA. In Figure 8 the adsorption isotherms for the five amino acids at pH 9 are presented. Adsorption isotherms for GLY at pH 7 and 11 were also measured, and it was found that the amounts of GLY adsorbed

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Jack et al.

Figure 9. Adsorption isotherms for GLY onto CHA (prepared without amino acid) at pH 7 (b) and 9 (2). The solid lines show the results of fitting the data to eq 3.

onto CHA at pH 11 were below the detection limit of the method used here (Figure 9). It should be noted that the concentration of amino acid used in the preparation of the particles shown in Figure 1 (i.e., in the presence of 1 M amino acid) lies in the plateau region (surface saturation) for all of the isotherms. However, the solutions used in the measurement of the ζ-potential titrations were necessarily diluted by a factor of 50, and equilibrium concentrations of amino acids are, therefore, approximately 0.02 M. The measured isotherms were all fitted to a Langmuir-type isotherm shown in eq 2

Γads Kc ) Γmax 1 + Kc

(2)

where Γads is the number of moles of adsorbed amino acid per unit surface area of CHA at a given equilibrium concentration (c) of amino acid in solution, Γmax is the maximum number of moles of amino acid that can be adsorbed (i.e., complete monolayer adsorption), and K is the affinity constant. The Langmuir isotherm assumes monolayer coverage, that there are no lateral interactions between adsorbed solute molecules, and that the surface is homogeneous with all adsorbing sites on the surface equivalent in energy. In reality, most surfaces are heterogeneous and molecules will tend to initially occupy lower energy surface sites, progressively occupying higher energy surface sites as the solute equilibrium concentration is increased. Nonetheless, the assumption of a Langmuir-type isotherm is often found to provide a useful method to determine equilibrium coverage and allow for comparisons of the affinity of adsorption between different but related systems. It should also be noted that the adsorption isotherms reported here were measured on CHA particles that were initially prepared without amino acid (i.e., the control samples in Figures 1-4), as described in the Experimental Section above, while the CHAamino-acid particles used in the previous analysis were precipitated in the presence of 1 M amino acid. Moreover, from the TGA and XPS measurements it is most likely that there is amino acid occluded within the particles shown in Figure 1, and it is not suggested that the formation of CHA in the presence of the amino acids is described by a Langmuir adsorption process. Instead, the adsorption isotherm measurements are presented here to determine if there is a correlation between the affinity of the amino acids and their effects on particle formation in the presence of amino acid and to better understand the ζ-potential titrations, which are equilibrium surface processes. These points are further discussed at the end of this section. The values for K and Γmax determined from the measured isotherms are shown in Table 4. It can be seen that the affinities

of the amino acid to the CHA surface at pH ) 9 are in the order of LYS > GLY > ALA > SER ≈ ASP, although all of the K values measured for the amino acids studied here span less than 1 order of magnitude (30-140 L mol-1). Moreover, this trend is qualitatively similar to that observed for the increase in surface potential at this pH (see Figure 4) and also for the degree of positive nature of the side amino acid at pH ) 9, which scales in the order of LYS (positive) > ALA, GLY (neutral) > SER (partial negative) > ASP (negative); see also the isoelectric points of the amino acids (pI) shown in Table 1. From Table 3 it can be seen that the surface Ca:P ratio measured by XPS (1.60) of the CHA control sample is lower than that of the bulk ratio (1.88), implying that the surface is either depleted in Ca2+ or rich in PO43-. It should be noted that the samples have been ‘washed’, as described above, prior to the XPS measurements to remove excess reactants. Similar changes in the surface Ca:P ratios of washed HA samples have been observed by XPS.59 Brown and Martin60 proposed that the origin of this effect is due to formation of thermodynamically stable calcium-deficient layers at the HA surface via a dissolution and reprecipitation mechanism. Such an effect is reported to be more pronounced for particles of high surface area, such as those prepared in this work, and in part responsible for the negative ζ-potential of the CHA particles observed at pH > 5. The adsorption affinities (K) of the amino acids measured here may, therefore, reflect an increased ease of approach of the more positively charged amino acids toward the negatively charged CHA interface. In addition, the relatively narrow range of K values determined between LYS and ASP, despite having opposite polarities in their side groups, suggests that the principle interaction between the amino acid and the CHA surface is not mediated through the side group. It should also be stated that the affinity constants measured for all of the amino acids investigated in this work are relatively weak. For example the equilibrium concentration of amino acids at one-half coverage on the isotherms, i.e., Γ/Γmax ) 0.5 (which is equal to K-1; see eq 2), are in the range of 7-30 mM. Typical values for high affinity adsorption are of the order of a few to tens of micromolar, e.g., adsorption of a specific peptide39 as well as catechol and related molecules30 onto HA and for adsorption of a cationic surfactant onto silicon.61 In addition, the affinity constants (K) determined in this work (30-140 L mol-1) are of a similar order of magnitude to the stability constants (Ksp) tabulated for the amino acid-Ca2+ complex;62 these range from 17-40 L mol-1. A number of previous authors have suggested that this interaction involves some form of coordination between the carboxyl groups and calcium ions at the surface of the HA or within the crystal,17,23-26 and such a mechanism is consistent with these observations at pH ) 9. From the XPS measurements of the CHA particles formed in the presence of the amino acids (Table 3) it can be seen that the ratio of Ca:P at the surface is either the same or slightly elevated with respect to the bulk. This finding suggests that the presence of the amino acid either suppresses depletion of calcium ions from the surface of the crystal during the washing process, as discussed above, or leads to depletion of phosphate ions. Previous studies on the adsorption of serine25 at pH 7 have suggested that there is a concurrent loss of PO43- from CHA upon adsorption of these molecules. However, in light of the previously discussed (59) Amrah-Bouali, S.; Rey, C.; Lebugle, A.; Bernache, D. Biomaterials 1994, 15, 269-272. (60) Brown, P. W.; Martin, R. I. J. Phys. Chem. B 1999, 103, 1671-1675. (61) Pereira, E. M. A.; Petri, D. F. S.; Carmona-Ribeiro, A. M. J. Phys. Chem. B 2006, 110, 10070-10074. (62) Martell, A. E.; Smith, R. M. Critical stability constants; Plenum Press: New York, 1974; Vol. I.

Modified Carbonate-Containing Hydroxyapatite Particles

finding that the amino acids stabilize particle growth by inhibiting the Ostwald ripening process, suppression of the surface depletion of Ca2+ from the amino-acid-functionalized surface is also considered likely to occur. Moreover, if the loss of phosphate alone was the main mechanism for the observed increase in the surface Ca:P ratio (cf. the CHA) then it would be likely that there would be a greater difference in the ratio for the differing amino acids and that these differences would reflect the different amounts of amino-acid adsorption seen in Figure 8 and the Γmax values determined. The increase in ζ-potential observed in Figure 4 upon addition of the amino acids is, therefore, due to a complex balance of any loss of PO43- and an increased retention of Ca2+ in addition to the positive charge associated with the -NH3+ terminus and any additional charge associated with the side group, which is enhanced in the case of LYS (positive) and reduced in the case of ASP (negative). The differences in the ζ-potentials of the CHA functionalized with the neutral amino acids (GLY, ALA, and SER) can then be further explained by considering the adsorbed amounts at an equilibrium concentration of ca. 0.02 M and pH ) 9, as shown in Figure 8. It can be seen that the SER and ALA isotherms show similar adsorbed amounts but are higher than that of the GLY. If adsorption is assumed to lead to loss of PO43- from the CHA crystal and, therefore, an increase in the solution concentration of PO43-, then from Figure 7 it can be seen that such an increase in the concentration of PO43- would lead to a reduction in the measured ζ-potential. Moreover, as the excess amino acids and ions in the solutions are in equilibrium with the surface, i.e. the surface amino acid is reversibly adsorbed onto the CHA surface, the decrease in ζ-potential observed for the GLY sample in decreasing pH from 9 to 7 is also consistent with the higher adsorbed amount of GLY at pH 7 (see Figure 9) and the subsequent increase in the solution concentration of PO43- upon this increased adsorption. Finally, the differences in the values of Γmax observed at pH 9 are not simple to explain in terms of molecular size or charge consideration. It can be seen from Figure 4 that the trends in the ζ-potential as a function of pH for the amino-acid-coated samples all follow the same general pattern. That is, they show a decrease in ζ-potential from pH 9.5, a plateau region at pH < ∼8 and then an additional decrease to a second plateau in the case of ASP, SER, and GLY, the onsets of which are dependent on the specific amino acid (pH ≈ 7, 6.3, and 6, respectively). Moreover, from Figure 6 it appears that there is a further decrease in ζ-potential for the GLY sample for pH > 10. The complex behavior observed in the ζ-potential titrations, i.e., multiple inflections and decreasing surface charge with increasing H+ concentration (decreasing OHconcentration), suggests that there may be a complex multisite adsorption process occurring, perhaps associated with different faces of the CHA crystal. The changing of pH may effectively ‘switch’ on or off certain sites for adsorption, and it is further likely that the nature of the interaction between the amino acid and the CHA (e.g., a surface complex between COO- and Ca2+) surface may be different at different sites. It is also interesting to note that the inflections occur at similar pH values to inflections observed in the CHA control, albeit with an opposite sign, and that the point of inflection at lower pH values (