Inhibition of Hydroxyapatite Formation by Zirconocenes - Langmuir

S. Koutsopoulos, Ch. Maniatis, C. D. Xenos, and E. Dalas. Crystal Growth & Design 2001 1 (5), 367-372. Abstract | Full Text HTML | PDF | PDF w/ Links...
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Langmuir 1995,lI , 1831-1834


Inhibition of Hydroxyapatite Formation by Zirconocenes S. Koutsopoulos, J. Demakopoulos, X. Argiriou, E. Dalas,* N. Klouras, and N. Spanos University of Patras, Department of Chemistry, GR-26500, Patras, Greece Received April 28, 1994. I n Final Form: February 16, [email protected] The effect of zirconocenes, used a s drugs for various therapeutic applications, on the crystal growth of hydroxyapatite was investigated at sustained supersaturation. All zirconocenes were found to inhibit crystal growth of hydroxyapatite possibily through adsorption onto the active sites for crystal growth. The kinetics results favored a Langmuir-type isotherm, suggesting a high affinity of zirconocene dichloride for hydroxyapatite.

Introduction Metallocene dihalides that possess antitumor properties are mainly represented by complexes of type RzMXz (X= F, C1, Br, I). These are organometallic complexes, the cyclopentadienyl ring ligands (R = C5H5, CH3C5H4) being bound t o the central atom M (M = Ti, Zr, V, Nb, Mo) by carbon-metal bonds,l as shown in Figure 1. The effectiveness of metallocene dihalides as antitumor agents varies with the position of the metal atom in the periodic table.2 Recently the antiarthritic activity of titanocene complexes has been m e a ~ u r e d . ~ These , ~ complexes are characterized by pronounced anti-inflammatory properties, givingrelief at optimum doses of 20-100 mgkg. The typical plasma level after administration was reported to be approximately 1 mM of RzMXZ.~ However, when applied in dimethylformamide o r dimethyl sulfoxide t o the skin, t h e y manifested both antiinflammatory and antiarthriticactivity without much local skin damage. They might therefore have t h e potential of being useful drugs, especially if released slowly. Despite the r a t h e r widespread use of metallocene dihalides as drugs, very little is known concerning their influence on t h e formation of the hard tissues of humans, composed mainly of basic calcium phosphate. Hydroxya p a t i t e (Ca5(PO&OH, HAP) is t h e model compound for the inorganic component of bones and teeth and was observed in pathological calcification of the articular In the present work we have investigated the effect of various zirconocenes dihalides which a r e listed i n Table 1. Their effect o n the crystal growth of HAP was studied by the constant composition a p p r ~ a c h . ~ - l l

Experimental Section The experiments were done at 37 k 0.1 "C in a thermostated double-walled,water-jacketedPyrexvessel, volume totaling0.250 dm3. Solid reagent-grade calcium chloride,potassium dihydrogen Abstract published in Advance A C S Abstracts, May 1, 1995. (1)Kopf-Maier, P.; Hesse, B.; Voitgtlander, R.; Kopf, H. J. Cancer Res. Clin. Oncol. 1980,97,31. (2)Dombrowski, K. E.;Baldwin, W.; Sheats, J. E. J. Organomet. Chem. 1986,302,281. (3)Fairlie, D. P.;Whitehouse, M. W.; Broomhead, J. A. Chem.-Biol. Interact. 1987,61, 277. (4) Dalas, E.; Klouras, N.; Maniatis, C. Langmuir 1992,8, 1003. (5)Toney, J. H.; Marks, T. J. J . Am. Chem. SOC.1985,107, 947. (6)Gordon, G. V.;Villanueva, T.; Shumacher, H. R.; Gohel, V. J . Reumatol. 1984,11, 861. (7)Gibilissco, P.A.;Schumacher, H. R.; Hollander, J. L.; Soper, K. A. Arthritis Rheum. 1985,28,511. (8) Boskey, A. L.; Bullogh, P. G. Scanning Electron Microsc. 1984, II, 943. (9)Koutsoukos,P.G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. SOC.1980,102, 1553. (10)Tomson, M. B.; Nancollas, G. H. Science 1978,200,1059. (11)Koutsoukos, P. G. Ph.D. Thesis, SUNYAB,1980. @

Figure 1. Zirconocene dichloride, a complex with a tilted sandwich structure. Table 1. Zirconocenes, Studied with Respect to Their Effect on the Crystal Growth of HYdrOXYaDatite zirconocene structurea zirconocene structurea ZF Zr(q5-C5H5)zFz MZC Zr(q5-C5H4CH3)2C12 ZC Zr(v5-C5H5)zC12 SMZC Zr(v5-C5H4SiMe3)2C1z SEZC Zr(i5-C5H4SiEt3)2Clz~ ZB Zr(i5-C5H&Brz ZI Zr(q5-C5H&Iz SBZC Zr(q5-C5H4But)zC12 ~



Me = CH3, Et = CH2CH3, But = t-C4H9.

phosphate, sodium chloride (Merck) and triply distilled C02free water were used in the preparation of the solutions. Potassium hydroxide solutions were prepared from concentrated standards (Merck, Titrisol). The standardization of the stock solutions prepared is described in detail elsewhere.9-11 The supersaturated solutions were prepared in a thermostated vessel by mixing the appropriate volumes of calcium chloride and potassium dihydrogen phosphate. The ionic strength of the solutions was adjusted to 0.15 mol dm-3 by the addition of sodium chloride. The solution pH was measured by a glass/saturated calomel pair of electrodes (Metrohm, 6.0101.100 and 6.0726.100, respectively) standardized before and after each experiment by NBS buffer solutions.12 Following pH adjustment, by the addition of dilute potassium hydroxide, the crystal growth process was initiated by the addition of 20 mg of well-characterized HAP seed crystals prepared by a method described e1~ewhere.l~ The specific surface area of the seed crystals, as determined by a multiple-point BET method (Perkin-Elmer sorptometer 212 D),was found to be 34.6 m2 g-l. The solid precipitates were analyzed by infrared spectroscopy (KBr pellet method, FTIR Perkin-Elmer 16-PC) and by powder X-ray diffraction (Philips PW 1830/1840)using aluminium as an internal standard and elemental analysis. The synthetic crystals displayed the characteristic powder X-ray diffraction patternl4 and the infrared spectrum (KBr pellet method, FTIR Perkin-Elmer 16-PC) of stoichiometric HAPll and the experimentally determined stoichiometric ratio Ca:P was 1.67 & 0.01. The zirconocene dihalides (q5-C5H&ZrX2with X = F, C1, Br, I were prepared and purified accordingto literature methods.15J6 (qW5H4SiMe3)~ZrClz was prepared as previously described17 and the remaining 1,l'-disubstituted zirconocene dichlorides (q5(12)Bates, R. G.In Determination o f p H , Wiley: New York, 1973. Koutsoukos, P. G.; Nancollas,G. H. J.Colloid Interface (13)Amjad, Z.; Sci. 1984,101,250. (14)ASTM File Card No. 9-432 (15)Samuel, E.Bull. SOC.Chim. Fr. 1966,11, 3548. (16)Druce, P.M.; Kingston, B. M.; Lappert, M. F.; Spalding, T. R.; Srivastava, J. Chem. SOC.(A) 1969,2106. (17)Kopf, H.; Klouras, N. Chem. Scr. 1982,19, 122.

0743-746319512411-1831$09.00/0 0 1995 American Chemical Society

1832 Langmuir, Vol. 11, No. 5, 1995

Koutsopoulos et al.

Table 2. Crystallization of HAP on HAP Seed Crystals in the Presence of Zirconocenes at pH 7.40,37 "C, 0.16 M NaC1, and Total Calcium (Cat):Total Phosphate (Pt) = 1.67 AGAJ mol-' exp

CaJ10-4 mol dm-3


3 2 4 7 8 9 11

12 13 14 19 18 15 20 21 22 50 30 31 32 33 29 P15 P16 nsc


5 5 5 5 5 5 5 5 5 5 5 5 5 4 3.5 3 2.5 5 4 3.5 3 2.5 5 5 5 5 5 5 5

zirc~nocene/lO-~ mol dm-3 ZFIO.78 ZB10.52 ZIl0.42 ZC10.68 MZClO.60 SMZCl0.49 SEZCI0.41 SBZCl0.30 ZC10.34 ZC11.03 zc11.71 ZC13.42 ZC16.84 ZCIO.68 ZC10.68 ZC10.68 ZC/O.68

HAP -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.0 -2.7 -2.0 -1.5 -3.5 -3.0 -2.7 -2.0 -1.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5 -3.5

x2 x3 a Hydrolysis time, 3 h. Hydrolysis time, 12 h. Hydrolysis time, 24 h. CsH4R)zZrClZ (R = Me, t-Bu, Et&) were obtained analogously. Elemental analyses (C, H, X) gave deviations 50.5% of the calculated values. The proton magnetic resonance and infrared spectra showed no evidence of impurities. In the HAP crystal growth experiments in the presence of zirconocene complexes, the latter were dissolved in the phosphate solutions along with the seed crystals, 30 min before the experiment started by the addition of calcium chloride. When following the above procedure, adsorption phenomena do not interfere with the kinetic measurements. Throughout the course of the crystallization process, water-saturated purified nitrogen was bubbled through the solutionin order to preclude atmospheric carbon dioxide from dissolving into the solution. During H A P formation, protons are released in the solution, thus offeringa very sensitive means of monitoring its formation. A pH-meter (Metrohm 691) was used for measuring pH. Connection of the pH-meter to a pH-stat (Metrohm 614 Impulsomat with 654 dosigraph) which was modified so as to accommodate two burets, mechanically coupled and mounted onto the shaft of the piston buret, allowed us, through the simultaneous addition of exactly equal volumes of reagents, to achieve invariability of all species in solution. This may be done by adding simultaneously calcium chloride-sodium chloride and potassium hydrogen phosphate-potassium hydroxide in a way such that the stoichiometry of the precipitating phase is preserved.1° At plethostatic conditions, therefore, the recorded motion of the buret piston can be easily translated into moles of HAP formed per unit time and area ofthe introduced seed crystals (R).However, owing to the reduction of the specific surface area during the crystallization process,lsthe rates were taken at titrant additions corresponding to precipitated HAP = 5%, with respect to the added H A P seed crystal concentration (20-45 min after the experiment started). Experiments with different amounts ofseed crystals (10, 15, and 20 mg) showed the same initial rates normalized per unit surface area of the substrate. Also, changes in the stirring rate (between 60 and 180 rpm) had no effect on the initial rates. It may therefore be suggested that crystal(18)Holl, H.; Koutsoukos,P. G.; Nancollas, G. H. J . Crystal Growth


TCP -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -0.6 -0.3 0.5 1.1

-1.2 -0.6 -0.3 0.5 1.1 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2 -1.2

DCPD 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.4 3.9 4.2 5.1 5.6 3.4 3.9 4.2 5.1 5.6 3.4 3.4 3.4 3.4 3.4 3.4 3.4

OCP 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.6 0.9 1.6 2.0 0.1 0.6 0.9 1.6 2.0 0.1 0.1 0.1 0.1 0.1 0.1 0.1

R/lO-s mol min-l m-2 2.63 1.71 2.89 2.46 4.21 4.62 5.68 3.34 3.41 1.42 1.21 0.99 0.57 1.38 0.98 0.79 0.44 9.72 5.38 3.85 3.04 1.68 9.69 9.20 9.90 10.05 2.0ga 1.12b 0.97e

lization took place e x ~ l u s i v e l yon ~~ the surface of the introduced seed crystals. Higher amounts of seed crystals lead to erroneous results because of the high ionic strength, 0.15 M NaC1, that may cause aggregation. The reproducibility of the measured rates of five controls (Table 2) was better than 5%. During the crystallization process, samples were withdrawn (0.5, 1, 3, 6, and 12 h after the experiment was started) and filtered through membrane filters (Sartorius,O.2pm). The filtrates were analyzed for calcium by atomic absorption and for phosphate by spectrophotometricmethods.20The constancyof calcium and phosphate was better than 3%. Electrophoreticmobility measurements of the apatite particles, both in the presence and in the absence of zirconocene dichloride, was performed in a microelectrophoresis apparatus (Rank Brothers MK 111, using a four-electrode cylidrical cell. The velocities of a t least 20 particles in each direction of the electric field were measured at the two stationary layers of the capillary tube.21 The measurements were made at constant ionic strength (0.01 mol dm-3) adjusted by sodium chloride. The pH of the suspensions was varied by adding small amounts of standard hydrochloric acid or sodium hydroxide, taking care to avoid any change of the ionic strength of the medium. Potentiometric titrations of zirconocene complexes were done a t 0.15 M NaCl ionic strength in the presence and the absence M at a constant temperature 37 "C. The oftotal calcium, 5 x aqueous suspensions were equilibrated for 1h before titration.22 Throughout the course of the experiments water-saturated purified nitrogen was bubbled through the solution in order to preclude atmospheric carbon dioxide from dissolving into the solution. The pH was recorded every 3 min as a function of the volume of titrant added to the suspensions. (19)Nyvlt, J.;Sohnel,0.;Matuchona, M.; Broul, M. The Kinetics of Industrial Crystallization; Elsevier: Amsterdam, 1985; pp 68, 284. (20) Reynolds, L.; Wilkinson, G. h o g . Nucl. Chem. 1969,9 , 86. (21) Overbeek, J. Th. G. In Colloid Science, Kruyt, H. R., Ed.;

Elsevier: Amsterdam, 1952; Vol. 1, p 219. (22) Vordonis, L.; Koutsoukos, P. G.; Lycourghiotis,A.J . Catul. 1986,

98. 296.

Inhibition of Hydroxyapatite Formation by Zirconocenes

Results and Discussion The driving force for the formation of a crystalline phase, MZ:XI (u = u+ + u-1, is the average change in Gibbs free

Langmuir, Vol. 11,No.5, 1995 1833 -

1 ti,

energy, AG, for the transition from the supersaturated solution to equilibrium and is given by

where the parentheses denote ionic activities and Tis the absolute temperature, R, is the gas constant, SZ is the supersaturation ratio, and KO, is the thermodynamic solubility product of the precipitating solid phase. A number of calcium phosphates may be formed in the supersaturated calcium phosphate solutions in order of increasing solubility: hydroxyapatite [HAP],tricalcium phosphate [CadPO4)2,TCPl,octacalcium phosphate [C%H(P04)3*2.5&0, OCP] and dicalcium phosphate dihydrate [CaHP04*2H20,DCPD]. The following values were used for the thermodynamic solubility products of the various calcium phosphates: for HAP, KO, = 2.35 x 10-59;23for for OCP, KO, = 5.01 x 10-50;25 TCP, KO, = 2.83 x 10-30;24 for DCPD, KO, = 1.87 x lo-’.? The computation of the activities of the free ions M*+ and Xz-was done a s previously de~cribed.~-ll In all cases the measured crystal growth rates, R , were proportional to the relative solution supersaturation, u, with respect to hydroxyapatite (5

= Q”9


R =k S 8







lno Figure 2. Kinetics of Wcrystallization on W seed crystals both in the presence ( 0 )and in the absence (A)of 0.68 x mol dm-3 Zr(y5-C5H5)Clza t pH’7.40, 37 “C, and 0.15 mol dm-3 NaC1. 1.6




where k is the precipitation rate constant, S a function of the active growth sites on the seed crystals, and n the apparent ordre of the reaction.27 Kinetics plots according to eq 3 gave a satisfactory fit, as may be seen in Figure 2. From the linear plots, avalue ofn = 2 f0.2 was obtained for the crystallization of HAP both in the presence and in the absence of chloride zirconocenes, which is higher than the value reported for the crystallization in low ionic strength media.11J3,24The value n = 2 is indicative of a surface-diffusion-controlled mechanism. The k S values were(l.2 f0.3) x 10-8and(0.32f0.02) x 10-8molmin-1 m-2 in the absence and in the presence of zirconocene dichloride, respectively. The experimental conditions and the kinetic results obtained are summarized in Table 2. The rate of crystal growth ( R ) was reduced upon increasing the solution concentration of zirconocene dichloride. Potentiometric titrations of the chloride zirconocene complexes under the conditions of the experiments (0.15 m NaCl), both in the presence and in the absence of total calcium, 5 x M, did not show any appreciable complexation, thus suggesting that the observed inhibitions are not due to a (23)Mc Dowel, H.; Gregory, T. M.; Brown, W. E. J. Res. Natl. Bur. Stand. (US.) 1977,81,273. (24)Gregzorv. T. M.: Moreno. E. C.: Patel, J. M.: Brown. W. E. J . Res. Natl. Bur. &and. (US.)1974,78, 667. (25)S h y , L. J.; Perez, L.; Zawacki, S. J.; Heughebaert, J. C.; Nancollas, G. H.J. Dent. Res. 1983,62, 398. (26)Marchall, R. Ph.D. Thesis, State University of New York at Buffalo, Buffalo, NY, 1970. (27)Nielsen, A. E.Pure Appl. Chem. 1981,53,2025. (28)Dalas, E.;Koutsoukos, P. G. J. Chem. SOC.Faraday Trans. 1 1989,85,2465. (29)Koutsoukos,P.G.;Amjad, Z.; Nancollas, G. H. J.ColloidInterface Scz. 1981,83,599. (30)Amjad, Z. Langmuir 1987,3,1063. (31)Maniatis, Ch.;Dalas, E.; Zafiropoulos, Th. F.; Koutsoukos, P. G. Langmuir 1991,7,1542.

1 I

.”n l


10 (l/C,)/lO‘



dm3 mol-’

Figure 3. Kinetics of H A P crystal growth in the presence of various concentrations of Zr(y5-C5H&C12, according to the Langmuir kinetic model a t pH 7.40, 37 “C, and 0.15 mol dm-3 NaC1.

decrease in solution supersaturation because of the Ca2+ sequestration by the zirconocenes. Thus the inhibition observed may be ascribed to further blocking of the active growth sites of the seed crystals. This assumption was tested by fitting the kinetics results in a Langmuir-type isotherm. It should be noted however that the basic assumptions on which the kinetic isotherm is based are that adsorption free energy is constant over the entire adsorbent surface and that there are no lateralinteractions between the adsorbed molecules. The rates of crystal growth in the absence, Ro,and in the presence, Ri,of the inhibitors may be related to their concentrations in the supersaturated solutions, Ci,according to

(4) In eq 4 k, and k d are the specific rate constants for the adsorption and desorption, respectively. The ratio (k$ k d ) , defined a s the “affinity constant”, may be determined from linear plots according to eq 4, like the plot in Figure 3. From the slope of the straight line, a value of 57.80 x lo4 mol cm-3 was obtained for the affinity constant of zirconocene, ZC. For comparison only, values of “affinity constants” for other inhibitors for the crystal growth of H A P are given in Table 3. The higher value of the “affinity constant”

Koutsopoulos et al.

1834 Langmuir, Vol. 11, No. 5, 1995 Table 3. Affinity Constants for Various Inhibitors of HAP Crystal Growth

io4 (k&d)

mol ~ I I ref~


phytic acid citric acid Mgz+ aminotris(methy1enephosphonic acid) 1-hydroxyethane-1,l-diphosphonic acid melitic acid glucose bis(su1fonamides) titanocenes Ti(q5-CsH~)2(Hz0)zZf zirconocene Zr(q5-CsH~)2(Hz0)22f a

8.4 1.5 1.54 62 208 160 10.22 3.51 68.88 57.80

29 30 13



30 29 30 28

31 4 a

This work.

points to the stronger adsorption of the inhibitor of the H A P surface. The cyclopentadienyl hydrolysis, initially follows approximate first-order kinetics, with a half-life of tu2 = 12.7 f 1.4h a t 37 “C and 0.103M NaC1.5 The amount of free cyclopentadiene in the working solution (50-75 min after zirconocene introduction into the working solution the crystal growth rate was measured) was insignificant. On the other hand, chloride hydrolysis resulted in a rapid increase of the hydrolysis products in the working solution, despite the presence of 0.15M NaC1.5 Zr(q5-C5H5),C1,

+ H,O



10.0 15.0 20.0 Hydrolysis time / h


Figure 4. The effect of the zirconocene hydrolysis products on the reduction of the rate of HAP crystal growth.


Z ~ ( T , I ~ - C ~ H ~ ) ~ ( H+, O C1-) C(~5+)


Z~(T,I~-C,H,)(H,O)C~+ H,O = Zr($-C5H5)(H,0),2+

+ C1-


The half-life for the first hydrolysis step of the ZC is very short (cannot be measured by chloride potentiometry) and the half-life for eq 6 is 31.7 min. Therefore, the creation of more positively charged centres via chloride hydrolysis is expected to lead to stronger interactions (electrostatic) between the negatively charged H A P and the positive sites of Zr(v5-C5H&(H20)C1+and Zr($-CsH&(HzO)~+ complexes. The hydrolysis of fluoride, bromide, and iodine zirconocenes produces additional anions of F-, Br-, and I-, respectively, that probably interfere in the inhibition procedure; consequently, only the “affinity constant’’ for the zirconocene dichloride was measured. The relative reduction of HAP crystal growth rates in the presence of zirconocene dichlorides was found to follow the trend blank =- MZC > ZC (Table 2). This may be due to the fact that the presence of the additional CH3 group decreases the positive charge of the chloride hydrolysis product. Finally, if the crystallization started 3,12,and 24 h after zirconocene chloride introduction (hydrolysis 1.122 x time) the rate measured was 2.09 x and 0.972 x mol min-l m-2, respectively, leading to the conclusion that there was a n additional effect of the zirconocene hydrolysis products, [Zr(v5-C5H5)2(H20)C1+, Zr(y5-C5H5)(H20)22+l on the inhibition of crystal growth, as shown in Figure 4. Since zirconocenes were shown to have a n appreciable effect on the crystallization of HAP, it is expected that it would also cause a change in the electrokinetic parameter of the HAP particles. In Figure 5 the electrophoretic mobility (EM) of the HAP particles in the presence and in the absence of zirconocene dichloride is shown. The







PH Figure 5. Electrophoretic mobility of HAP particles in 0.01 mol dm+ NaCl at 37 “C (A)in the absence of any additive and (0)in the presence of 0.68 x mol dm-3 Zr(y6-C5H5)zC12.

electrokinetic charge of HAP followingadsorption is shifted to more negative values.

Conclusions In the present work, the effect of zirconocene complexes on the crystallization of HAP, in the concentration range to 6.84x mol dm-3 was investigated from 0.34x in solutions supersaturated only with respect to HAP, a t constant solution composition. The zirconocene dihalides investigated reduced the rates of crystal growth of HAP by 42-95%. The inhibitory effect may be explained by the adsorption and subsequent blocking of the active growth sites. The adsorption assumption may be justified through the satisfactory fit of the results to a kinetics Langmuir-type isotherm. Acknowledgment. We acknowledge the partial support of the Ministry of Energy and Technology of the Greek Government, Grant EA 857. LA9403542