on the Crystal Growth of Hydroxyapatite - ACS Publications - American

Chemical Engineering and the Institute of Chemical Engineering and Chemical Processes at. High Temperatures, P.O. Box 1239, Patras, Greece. Received J...
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Langmuir 1991, 7, 1542-1545


Effect of Various Bis(su1fonamides) on the Crystal Growth of Hydroxyapatite Ch. Maniatis,+E. Dalas,? Th. F. Zafiropoulos,t and P. G. Koutsoukos’J Department of Chemistry, University of Patras, GR-26110, Patras, Greece, and Department of Chemical Engineering and the Institute of Chemical Engineering and Chemical Processes at High Temperatures, P.O. Box 1239, Patras, Greece Received July 24,1990. I n Final Form: December 20, 1990 The effect of four bis(sulfonamides),N,”-bis(2-nitrobenzenesulfonyl)- 1,2-diaminoethane(BSl),N,”bis(4-nitrobenzenesulfonyl)-1,3-diaminopropane (BS2), N,”-bis( 2-aminobenzenesulfony1)-1,Bdiamino(BS4), used as drugs for various ethane (BS3), and N,N’-bis(4-nitrobenzenesulfonyl)-l,2-diaminoethane therapeutic applications, on the crystal growth of hydroxyapatite was investigated at sustained supersaturation. All bis(su1fonamides)were found to inhibit crystal growth of hydroxyapatite possibly through adsorption onto the active sites for crystal growth. The order of crystal growth rates was found to be as follows: blank > BS3 = BS2 > BS1= BS4. This behavior was ascribed to the electron-withdrawalcharacter of the NO1 groups, which seemed to favor adsorption. The kinetics results favored a Langmuir-type isotherm, suggesting a high affinity of BS4 for hydroxyapatite.

Introduction Sulfonamides are drugs of proven therapeutic importance and are used against a wide spectrum of bacterial ailments,l12in the treatment of cancer? malaria: leprosy: and tuberculosis! and for the inhibition of human carbonic anhydrase II.’ Recently, a number of sulfonamide metal complexes of importance in controlling various pathogen fungi819has been studied due to the existing evidence of their biological activity. Since their introduction, 40 years ago, the use of sulfonamides has been increasingly widespread because a variety of them are available with markedly different adsorption and excretion rates. Moreover they are easily adsorbed and they do not interfere with host defense mechanisms. Sulfonamides along with sulfones exhibit moderate toxicity with oral lethal dose of 0.5-5 g/kg for humans. Typical peak plasma levels 6 days after administration are reported to have reached a level of 0.03 pg/mL, which was maintained for about 60 days. Reaching higher levels is also possible and typical half-lives reported are 43 days, although large variations may occur depending on the type of the living organism administered.lOJ1 Despite the rather widespread use of sulfonamides as drugs, very little is known concerning their influence on the formation of the hard tissues of humans, composed mainly of basic calcium phosphate. Hydroxyapatite (Cas(PO&OH, HAP) is the model compound for the + Department of

Chemistry, University of Patras.

1 Department of Chemical Engineering and Institute

of Chemical

Engineering and Chemical Processes at High Temperatures. (1) Clayson, D. B.; Pringle, J. A. S.; Ranses, G. M. Biochem. Pharmacol. 1967,16, 1967. (2) Beerlev, W. N.; Peters, W.; Mager, K. Ann. Trop. Med. Parasitol.

1960,62, 288. (3) Hoffman LaRoche & Co., Swiss Patent, 416648, 1967. (4) Schmidt, L. H. Annu. Reu. Microbiol. 1969,23,427. (5) Tarbini, G. Proc. Znt. Cong. Chemother. 1967,2, 909. (6) Vaichulis, J. A. U.S. Patent 3,272,352, 1966. (7) Vedani, A.; Dunitz, J. D. J. Am. Chem. SOC.1986,107, 7653. (8) Perlepes, S. P.; Zafiropoulos,Th. F.; Galinos, A. G.; Tsangaris, J. M. Chem. Scr. 1983,22, 226. (9) Gavioli, G. B.; Menabue, L.; Saladini, M.; Sola, M.; Corradi, A. B.; Battagha, L. P. J. Chem. SOC.,Dalton Trans. 1989, 1345. (10) Amand, N. Antibiotics; Corcoran,J. W.,HahnF.E.,Eds.;Springer Verlag: New York, 1975; pp 668-698. (11) Gosselin,R.E.;Hodge,H.C.;Smith,R.P.;Gleason,M. N. Clinical Toxicology of Commerical Products, 4th ed.; Williams & Wilkins: Baltimore, MD, 1974; pp 3-4, 77-86, 238.

inorganic comDonent of bone and teeth. In the Dresent workY we have-investigated the effect of various bis(su1fonamides), which are listed in Table I. Sulfonamides have a relatively low solubility in water, so we have investigated their effect at concentrations up to about 15 pg/mL, which was the limit of their solubility. The effect on the crystal growth of HAP was studied by the constant solution composition approach.12J3

Experimental Section All experiments were performed at 37.0 f 0.1 O C in a thermostated double-walled, water-jacketed Pyrex vessel, volume totalling 0.200 dm3. Solid reagent-grade calcium chloride, potassium dihydrogenphosphate,sodium chloride (Merck),and triply distilled, COz-free water were used in the preparation of solutions. Potassium hydroxide solutions were prepared from concentratedstandards (Merck,Tritrisol). The standardization of the stock solutionsprepared is described in detailel~ewhere.~zl~ The supersaturatedsolutionswere prepared in the thermostated reactor by mixing equal volumes of calcium chloride and potassium dihydrogen phosphate. The ionic strength of the solutionswas 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 (Radiometer, C202G and K402 respectively),standardized before and after each experimentby NBS buffer solution^.^^ Following pH adjustment, by the additionof dilute potassiumhydroxide,the crystalgrowth process was initiated by the addition of known quantities of wellcharacterizedHAP seed crystalsprepared by a method described else~here.’~J~ The specific surface area of the seed crystals, as determined by a multiple-point BET method (Perkin-Elmer spectrometer 212D), was found to be 34.6 m* gl. The solid precipitates were analyzed by infrared spectroscopy and by powder X-raydiffractionusing aluminum as an internal standard. The synthetic crystals displayed the characteristicpowder X-ray diffraction pattern” and the infrared spectrum (KBr pellet method, Perkin-Elmer 577 infrared spectrometer) of stoichiometric HAP.16 The stoichiometric ratio Ca:P experimentally determinedwas 1.67 0.01. Bis(su1fonamides)weresynthesized by the method described elsewheres and were characterized by the melting point, elementalanalysisfor C, H, and N, and infrared (12) Koutsoukos, P. G.; Amjad, Z.; Tomson, M. B.; Nancollas, G. H. J. Am. Chem. SOC.1980,102,1553. (13) Tomson, M. B.; Nancollas, G. H. Science 1978,200,1059. (14) Bates, R. G. In Determination of pH; Wiley: New York, 1973. (15) Koutsoukos, P. G. Ph.D. Thesis, SUNYAB, 1980. (16)Koutsoukos,P.G.; Amjad, 2.;Nancollas, G. H. J. Colloidlnterface Sci. 1981, 83 (2), 599. (17) ASTM File Card 9-432.

0743-7463/91/2407-1542$02.50/0 0 1991 American Chemical Society

Hydroxyapatite Crystal Growth

Langmuir, Vol. 7,No. 7,1991 1543

Table I. Bis(sulfonamides), Studied with Respect to Their Effect on the Crystal Growth of Hydroxyapatite bis(sulfonname amide) structure BS1 S02.NH(CHJ$4H-S0, N,”-bis(2-nitrobenzenesulfonyl)NO^ [email protected] 1,a-diaminoethane BS2

0 0 0 SO,-NH(CH&NH-SOz NHz










@J OzN

spectroscopy. The results of the above analysis were in excellent agreement with the theoretically expected values.8 In the HAP crystal growth experiments in the presence of sulfonamides, the latter were dissolved in the supersaturated solutions. Throughout the course of the crystallization process, water-saturated, purified nitrogen was bubbled through the solution in order to preclude atmospheric carbon dioxide from dissolving into the solution. Following the introduction of HAP seed crystals into the supersaturated solutions, crystallization began immediately and the solution supersaturation was kept constant by the addition of titrant solutions. Two mechanically coupled burets were used with the following composition: buret 1,(10 Cat 2 Cat) M Ca(N03)~; buret 2, (10Pt + 2 Pt) M KHzPO, + KOH; with molar ratios of Cat:Pt:OH = 5:3:1. The monitoring of the crystal growth process and the constant supersaturation approach have been described in detail in other publication~.~~J3 Experiments done both in the presence and in the absence of bis(su1fonamides)at different seed concentrations showed that crystallization took place exclusivelyon the surface of the introduced seed crystals. The reproducibility of the measured rates was better than 5 % (a mean of five experiments). During the crystallization process, samples were withdrawn and filtered through membrane filters (Millipore, 0.22 pm). The filtrates were analyzed for calcium by atomic absorption methods and for phosphate by spectrophotometric methods.l* The electrokinetic measurements were done by microelectrophoretic mobilitymeasurements using a Rank Brothers microelectrophoresis instrument, Model MKII, equipped with a capillary cylindrical four-electrode cell. Twenty particles were measured in each direction of the electric field a t each of the two depths where the two stationary layers are located. The measurements were done at constant ionic strength (0.15 mol dm-3) adjusted by sodium chloride at 37 “C.


Results and Discussion The experimental conditions and the kinetic results obtained are summarized in Table 11. A number of calcium phosphates may be formed in the supersaturated calcium phosphate solutions in the following order of increasing solubility: HAP, tricalcium phosphate [Ca3(PO4)2,TCP], octacalcium phosphate [CaH(P04)3.2.5HzO,OCP], and dicalcium phosphate dihydrate [CaHP0~2H20,DCPD]. The driving force for the formation of a crystalline phase, Mz+,+Xz-,- (u = u+ + u-) is the average change in Gibbs free energy, per ion, AG,for the transition from the supersaturated solution to equilibrium and is given by

(18)Murphy, J.; Riley, J. P. Anal. Chim. Acta 1962,27, 31.




Lnu Figure 1. Kinetics of crystallization of HAP seed crystals: pH 7.40, 37 OC, 0.15 mol dm-3 NaCl.

where the parentheses denote ionic activities, T is the absolute temperature, R, is the gas constant, fl is the superstation ratio, and KO, the thermodynamic solubility product of the precipitating solid phase. The following values were used for the thermodynamic solubility products of the various calcium phosphates: for HAP, KO, = 2.35 X 10-69,19 for TCP, KO,= 2.83 X 10-30,20for OCP, K O , = 5.01 X 10-w,21and for DCPD, K O , = 1.87 X 10-7.22The computation of the activities of the free ions Mz+and Xzwas done as previously In all cases, the measured crystal growth rates, R, were proportional to the relative solution supersaturation, u, with respect to hyroxyapatite Q

= Q’P - 1

R = kSu”



where k is the precipitation rate constant, S a function of the active growth sites on the seed crystals, and n the apparent order of reaction. Kinetics plots according to eq 3 gave a satisfactory fit, as may be seen in Figure 1. From the linear plots, a value of n = 2 was obtained for the crystallization of HAP, which is higher than the value reported for the crystallization in low ionic strength media.15326 Potentiometric titrations of bis(sulf0namides) at the concentrations of the experiments both in the presence (19) McDowel, H.; Gregory, T. M.; Brown, W.E. J. Res. Natl. Bur. Stand., Sect. A 1977,81, 273. (20) Gregory, T. M.; Moreno, E. C.; Patel, J. M.; Brown, W.E. J. Res. Natl. Bur. Stand., Sect. A 1974, 78, 667. (21) Shyu,L. J.; Perez, L.; Zawacki, S. J.; Heughbaert,J. C.; N a n c o h , G. H. J. Dent. Res. 1983,62, 398. (22) Marshall,R. PhD. Thesis,StateUniversity of New York at Buffalo, Buffalo, NY,1970. (23) Nancoh, G. H.; Amjad, Z.;Koutaoukos, P. G. In Chemical Modelling in Aqueous System; ACS Symp. Ser., No. 93; Jenne, E., Ed.; American Chemical Society: Washington,DC, 1980; p 475. (24) Nancolla~.G. H. Interactions in Electrolyte Solutions:Eleevier: &&dam, 1”. (25) Davies, C. W.Ion Association; Butterworthe: London, 1962. (26) Dalas, E.; Koutaoukos, P. G. J. Chem. SOC., Faraday Trans. 1 1989, 86 (lo), 3159.

1544 Langmuir, Vol. 7, No. 7, 1991

Maniatis et al.

Table 11. Crystallization of HAP on HAP Seed Crystals in the Presence of Bb(rulfonamides),B S pH 7.40,37 OC, 0.15 M NaCl, Total Calcium Cat:Total Phosphate Pt = 1.67 AG/kJ mol-' expt no. Cat/lo-' mol dm-* B S / W mol dm-* HAP TCP OCP DCPD R110-8 mol min-1 m-2 241 5.0 BS4 11.14 -3.5 -1.2 6.32 0.1 3.4 0.1 242 -3.5 5.0 -1.2 3.4 B Sj3.48 ~ 3.31 5.0 -3.5 0.1 3.4 BS412.32 5.06 243 -1.2 5.0 -1.2 -3.5 244 0.1 3.4 BS412.90 4.21 -3.5 5.0 -1.2 0.1 BS410.58 245 3.4 8.00 0.1 5.0 -1.2 BS411.16 246 -3.5 6.14 3.4 0.1 231 5.0 -1.2 BS311.35 -3.5 8.42 3.4 0.1 BSlf1.12 211 5.0 -1.2 -3.5 7.16 3.4 0.1 -3.5 221 5.0 -1.2 3.4 BS211.30 8.00 5.0 -3.5 0.1 3.4 29 -1.2 9.69 0.1 5.0 -1.2 -3.5 3.4 9.20 P15 0.1 5.0 -3.5 -1.2 3.4 9.90 PI6 -1.2 nsc 5.0 0.1 10.05 -3.5 3.4 -3.5 -1.2 3.4 9.13 50 5.0 0.1 -3.0 -0.6 5.39 0.6 30 4.0 3.9 -2.7 31 3.5 0.9 -0.3 4.2 3.84 3.0 -2.0 0.5 1.6 5.1 3.03 32 2.5 -1.5 2.0 1.69 1.1 33 5.6 Table 111. Affinity Constants for Various Inhibitions of HAP Crystal Growth

(k.lkd) x lo4 8.4 1.5 1.54 62 208

inhibitor phytic acid citric acid Mg2+


melitic acid glucose bis(sulfonamide) (BS4)

ref 16 28 29 28 16 28 30 this work

160 10.22 3.51




(l/ci)/ 104dm3 mol-'


Figure 2. Kinetics of HAP crystal growth in the presence of various concentrationsof bis(su1fonamide)BS4 according to the Langmuir kinetic model: pH 7.40, 37 "C, 0.15 mol dm-* NaCl. and in the absence of Ca2+(5 X 10-4M)did not show any appreciable complexation, thus suggesting that the observed inhibition is not due to a decrease in solution supersaturation because of the Ca2+ sequestration by the bis(su1fonamides). As may be seen from Table 11, bis(sulfonamide) BS4 showed the strongest inhibitory effect on the crystal growth of HAP. The inhibition observed may be ascribed to adsorption and subsequent blocking of the active growth sites of the HAP seed crystals. This assumption was tested by fitting the kinetics results in a Langmuir-type kinetic i~otherm.~'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 (27) Davies, C. W.; Nancollas, G. H. J. Chem. SOC.191111,818.









Figure 3. Electrophoreticmobility of HAP particles in 0.15 mol in the dm-*NaC1,37 O C : (0)in the absence of any additive; (0) presence of 3.48 x 1od mol dm-* bis(sulfonamide)BS4. adsorbent surface and that there are no lateral interactions between the adsorbed molecules. The results in Table I1 clearly show that the rate of HAP crystal growth was reduced upon increasing the solution concentration of bis(sulfonamides), a fact that may be ascribed to further blocking of the active growth sites. The rates of HAP crystal growth in the absence, Ro, and in the presence, Rip of the inhibitors may be related to their concentrations in the supersaturated solutions, ci,

Langmuir, Vol. 7, No. 7, 1991 1545

Hydroxyapatite Crystal Growth according to

R, R, - R~= 1




(k,) ci


In eq 4 k, and k d are the specific rate constants for the adsorption and desorption, respectively. The ratio (k,/ kd),defined as the "affinity constant" may be determined from linear plots according to eq 4, like the plot shown in Figure 2. From the slope of the straight line, a value of 3.51 X lo4 was obtained for the affinity constant of bis(sulfonamide) BS4. For comparison, values of affinity constants for other inhibitors for the crystal growth of HAP are given in Table 111. The relative reduction of HAP crystal growth rates was found to follow the trend blank > BS3 = BS2 > BS1 = BS4. A clear conclusion from the kinetics data is that the inhibitory effect of the -NH2 containing sulfonamides was stronger as compared to that of the -NO2 containing compounds. This may be due to the fact that the electrondrawing NO2 groups tend to increase the positive charge on the -SO2 groups of the aromatic nucleus, contrary to the effect of NH2 groups, which tend to make these groups more negative. The creation therefore of more positively (28) Amjad, Z. Langmuir 1987, 3, 1063. (29) Amjad,Z.;Koutsoukos,P.G.;Nancollas,G. H.J. Colloidlnterface Sci. 1984, io1 (I), 250. (30) Dalae, E.; Koutsoukoe,P. G.J . Chem. Soc., Faraday Trans. 1989.

85 (81,2465.

charged centers by the presence of NO2 groups on the aromatic nucleus is expected to lead to stronger interaction (electrostatic) between the negatively charged HAP and the positive sites of the NO2 containing bis(sulf0namides). Indeed, as may be seen in Figure 3,the interaction of the BS4 sulfonamide with HAP resulted in a shift of the isoelectric point of HAP from pH 6.4 to about pH 7.0, rendering a t the same time the HAP surface more positive. The position of the groups with respect to the -SO2 group (ortho or para) did not influence significantly the rates of precipitation.

Conclusions In the present work, the effect of bis(sulfonamides) on the crystallization of HAP, in the concentration range 0.58 X to 3.48 X 10+mol dm-3, was investigated in solutions supersaturated only with respect to HAP, a t constant solution composition. The bis(sulfonamides)investigated at concentrations in the range between 6 and 35 rcM reduced the rates of crystal growth of HAP by 17-65 % This inhibitory effect may be explained by 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. Finally it should be noted that the strongest inhibition was exhibited by the bis(sulf0namides), which contained the electron-drawing NO2 groups.