Biomaterials 26 (2005) 2073���2080 Novel biomaterials for bisphosphonate delivery Solen Jossea,Corinne Faucheuxb,A. Soueidanb,Ga. el Grimandib,Dominique Massiotc, Bruno Alonsoc,Pascal Janviera,Samia La. ��b a,Paul Piletb,Olivier Gauthierb,Guy Daculsib, Jerome # Guicheuxb,Bruno Bujolia,*,Jean-Michel Boulerb,1 a Faculte �� des Sciences et des Techniques, Laboratoire de Synth " ese Organique, University of Nantes, UMR CNRS 6513 and FR CNRS 2465, 2 Rue de la Houssini " ere , BP92208, 44322 Nantes Cedex 3, France b Faculte �� de Chirurgie Dentaire, Materiaux �� d���Inter �� # et Biologique, EM INSERM 99-03, BP84215, 44042 Nantes Cedex 1, France c CRMHT, UPR CNRS 4212, 1D Avenue de la Recherche Scientifique, 45071 Orleans �� Cedex 02, France Received 23 January 2004 accepted 26 May 2004 Available online 2 July 2004 Abstract One type of gem-bisphosphonate (Zoledronate) has been chemically associated onto calcium phosphate (CaP) compounds of various compositions. For that purpose,CaP powders of controlled granulometry have been suspended in aqueous Zoledronate solutions of variable concentrations. Using mainly 31P NMR spectroscopy,two different association modes have been observed, according to the nature of the CaP support and/or the initial concentration of the Zoledronate solution. b-tricalcium phosphate (b- TCP) and mixtures of hydroxyapatite and b-TCP (BCPs) appear to promote Zoledronate-containing crystals formation. On the other hand,at concentrations o0.05moll 1 CDAs (calcium deficients apatites) seem to undergo chemisorption of the drug through a surface adsorption process,due to PO3 for PO4 exchange,that is well described by Freundlich equations. At concentrations 0.05mol l 1,crystalline needles of a Zoledronate complex form onto the CDAs surface. The ability of such materials to release Zoledronate,resulting in the inhibition of osteoclastic activity,was shown using a specific in vitro bone resorption model. r 2004 Elsevier Ltd. All rights reserved. Keywords: Apatite structure Calcium phosphate Drug release Osteoclast 1. Introduction ������Smart������ methods of local-delivering drugs could reduce side effects,improve efficacy of existing drugs and open the door to entire classes of new treatments. Such delivery systems are able to precisely control the timing of a drug release by adjusting the properties of the carriers. Synthetic polymers are widely used as carriers [1] since they do not cause any significant inflammation to tissues at the implantation site. In that case,the rate of drug release can be controlled by various mechanisms: diffusion out of the matrix that remains intact,simultaneous drug release and degrada- tion of the matrix,or drug expulsion by osmotic pressure [2]. The incorporation of biomolecules into inorganic materials,such as silica gel mainly obtained by sol���gel methods [3���10] has also been extensively studied [11]. In this context,calcium phosphate ceramics (CaPs), commonly used as implants for bone reconstruction [12���18] appear to be also good candidates as biocompa- tible carriers,since they can be resorbed by cells and they promote new bone formation by releasing calcium ions and phosphates. Various CaPs-based drug delivery systems have been developed,consisting either of porous ceramic sealed reservoirs filled with the drug or CaP/ drug mixtures compressed as pellets or granules [19���23]. The purpose of our work was to chemically combine CaPs with geminal bisphosphonates,usually called bisphosphonates (BPs) which are used for the treatment of post-menopausal osteoporosis [24]. Indeed,potential complementarities appear between BPs and calcium phosphate materials for bone consolidation in main osteoporosis-induced fracture sites (femur neck,distal radius,vertebral bodies). The local release of BPs could ARTICLE IN PRESS *Corresponding author. Fax: +33-251-125-402. E-mail addresses: bujoli@chimie.univ-nantes.fr (B. Bujoli),jmbou- ler@sante.univ-nantes.fr (J.-M. Bouler). 1 Also for correspondence. 0142-9612/$- see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2004.05.019

be an interesting alternative to the oral administration, often suffering of low bioavailabilility and showing noticeable gastrointestinal disturbances [25���26]. More- over,we aimed at determining whether this device could favor the osseous integration of metallic implants (such as hip prostheses) within osteoporotic sites. Denissen et al. [27���31] have reported the biological properties of hydroxyapatite (HA) monolithic implants incubated in 1-hydroxy-3-dimethylamino-propylidene-bisphosphonic acid (olpadronate) solutions in dental surgery,but however,no data regarding the nature of the HA���BP chemical association was reported. On the other hand, as pure hydroxyapatite dissolves too slowly,it is of little practical interest for bone substitute applications [32]. For these reasons,we have developed bioactive implants based on more soluble CaPs,such as b-TCP (b���Ca3PO4),BCPs (biphasic calcium phosphates: a mixture of HA and b-TCP) or CDAs (calcium deficient apatites,Ca10 x(PO4)6 x(HPO4)x(OH)2 x),leading to injectable bone substitutes [33���35] showing an adapted balance between material resorption and new bone formation [36,37]. In order to maximize the expected therapeutic effect, we selected 1-hydroxy-2-(imidazol-1-yl-amino)-ethyli- dene-bisphosphonic acid (Zoledronate) (Fig. 1),a nitrogen-containing bisphosphonate with high anti- resorptive potency,identified as the most potent inhibitor of farnesyl diphosphate synthesis and inducer of osteoclastic apoptosis [38]. In the present study,we describe the association of these CaPs with Zoledronate along with the physical and biological properties of the resulting materials. 2. Experimental 2.1. Materials and methods CDA(NH3) (Ca=P �� 1:56) was obtained by basic hydrolysis of 320 g of dicalcium phosphate dihydrate (DCPD),using a mixture of 440ml of a molar solution of aqueous ammonia and 3.57 l of deionized water (4 h at 80 C). CDA(NaOH) (Ca=P �� 1:57) was prepared as previously described [36���37]. The Ca/P ratio of the samples was controlled on a Philips PW 1830 diffract- ometer,from the X-ray diffraction powder pattern of the corresponding calcined phases,according to the literature [39]. BCPs were prepared by calcination of CDA powders at 1050 C (4 h) in air. The desired HA/b-TCP ratios were tuned by adjusting the Ca/P ratio of the starting CDA (i.e. pure b-TCP from CDA(NaOH) with Ca=P �� 1:5 BCP(HA25-TCP75) from CDA(NaOH) with Ca=P �� 1:54 BCP(HA75-TCP25) from CDA(NaOH) with Ca=P �� 1:63) [32,40]. The grain size of all the CaPs used in this study was adjusted to 40���80 mm. Zoledro- nate was a gift from Novartis Pharma Research. The determination of the phosphorus content in solution [40] was performed using a Shimadzu UV- 160A UV-visible spectrometer. The scanning electron microscopy (SEM) experiments were carried out on a JEOL 6400F microscope. The 31P NMR spectra, recorded in solution,were taken on a Bruker AC 200 spectrometer,with NaH2PO4 as an external standard (capillary tube insert). The solid-state 31P NMR spectra were obtained on a Bruker DSX 300 spectrometer with 85% H3PO4 as the reference. 31P{1H}CP spectra were recorded at a MAS frequency of 10kHz,using a variable amplitude CP pulse program,a contact time of 1 ms,recycle delays of 1 or 2s,and a pulse RF field strength of about 50kHz. The particle size was measured using laser granulometry on a Coulter LS230 apparatus (Miami,USA). The sodium and calcium concentrations in solution were determined using a Unicam 989 atomic absorption spectrometer (Cambridge,UK). The carbon and nitrogen content of the Zoledronate-loaded CaPs was determined by che- mical analyses performed by the CNRS Analysis Laboratory (Vernaison,France). 2.2. Zoledronate loadings onto the CaPs The loading of bisphosphonate was performed using (i) CDAs obtained by hydrolysis of dihydrated dical- cium phosphate with an aqueous NaOH solution (CDA(NaOH)) or aqueous ammonia (CDA(NH3)),(ii) b-TCP,and (iii) BCPs obtained by thermal decomposi- tion of CDAs,the Ca/P ratio of which allows tuning of the composition (HA/b-TCP ratio) of the resulting BCP. Zoledronate loading onto the CaPs (40���80 mm) was performed by reacting the solid with an aqueous solution of the drug. Typically,a suspension of the desired calcium phosphate precursor (200 mg) in 1ml of an ultrapure water (18 M O cm) solution of zoledronic acid (the disodium form) of the desired concentration (see Table 1),was placed in an assay tube that was rotated slowly and mechanically (16 rpm) for 48h. After centrifugation,the solid was filtered and washed 4 times ARTICLE IN PRESS NaHO3P PO3HNa OH N N Fig. 1. Chemical structure of Zoledronate. S. Josse et al. / Biomaterials 26 (2005) 2073���2080 2074