Bioactive Glasses Loaded With Dexamethasone by a Supercritical Carbon Dioxide Deposition Process L.T.R. Filipe1, A.P. Piedade2, J.J. Watkins3, M.E.M. Braga1, H.C. de Sousa1* 1 CIEPQPF, Chemical Engineering Department, FCTUC, University of Coimbra, Rua Sílvio Lima, Pólo II – Pinhal de Marrocos, 3030-790 Coimbra, Portugal. 2 CEMUC, Mechanical Engineering Department, FCTUC, University of Coimbra, Rua Luís Reis Santos, Pólo II Pinhal de Marrocos, 3030-788 Coimbra, Portugal. 3 Department of Polymer Science and Engineering, University of Massachusetts, 120 Governors Drive, Amherst, MA 01003, USA. Corresponding author: [email protected]; Phone: (+351) 239 798 749; Fax: (+351) 239 798 703 ABSTRACT Bioactive glasses (BGs) have been quite used in implants, fixation devices and hard tissue engineering applications mostly due to their chemical and physical properties as well as to their bioactivities which allow the development of an apatite-like layer when in contact with physiological fluids. These inorganic materials are usually prepared by melting and quenching but they can also be prepared using sol-gel methods. In this work we prepared BGs (based on SiO2, P2O5, Na2O, CaO) and silica-calcium hydroxyapatite (HA) composites, by a onepot aqueous sol-gel method (at 37 ºC and atmospheric pressure) and using two biomimetic catalysts for the silica formation from tetraethyl orthosilicate (TEOS): L-glutathione-reduced (GSH) and cysteamine (CYS). Other precursors included triethyl phosphate (TEP), calcium nitrate tetrahydrate (CaNT) and sodium nitrate (NaNO3). Additional samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic calcium hydroxyapatite or by changing the employed amount of water. In addition, some samples were also obtained by adding chitosan (CHIT) as a porogenic agent. After synthesis, all obtained materials were dried, aged and submitted to a specific sinterization process (from 60 ºC up to 1000ºC). Obtained materials were characterized by mercury intrusion porosimetry, nitrogen adsorption, helium pycnometry, simultaneous differential thermal analysis (SDT), scanning electron microscopy (SEM), infra-red spectroscopy (FTIR) and X-ray diffraction. Obtained materials were later loaded with dexamethasone (DXMT, a hydrophobic drug with osteogenic properties) by a supercritical carbon dioxide (scCO2) deposition process or by conventional loading from aqueous solutions. Drug loading yields were changed by controlling scCO2 densities (755-920 Kg/m3) at a constant depressurization rate (0.5 MPa/min) and at constant processing times (14 hours). Loading yields and drug release profiles were obtained and compared to those obtained by the conventional method. The obtained results, in terms of porosity, surface morphology and drug release profiles, demonstrated the feasibility of the employed sol-gel and supercritical methods for the development of DXMT-loaded composite materials with potential uses for pharmaceutical and biomedical applications. INTRODUCTION In recent years, bioactive glasses (BGs) have been used as important biomaterials in implants, fixation devices and other hard tissue engineering applications [1-3]. The most used and best characterized BG is type 45S5, which is a silicate glass that contains 45% SiO2, 24.5% NaO2, 24.5% CaO and 6% P2O5 (w/w) [1] and that has been already employed in several FDA-approved implants and devices [2]. BGs present favorable and adequate chemical, physical and mechanical properties as well as high bioactivities which will induce the development of a surface apatite-like layer when in contact with physiological fluids [1, 3-6]. This will promote enhanced in vivo bone bonding and integration without causing inflammation, toxicity or forming fibrous tissues [1, 3]. Once the layer is formed, a firm connection between BGs and the bone structure (host bone) is allowed, owing to the similar mineral composition from the layer and bone and to the leaching of Na+ and Ca2+ cations to the surrounding fluids [1, 3, 4]. These inorganic materials are usually prepared by melting and quenching methods despite the fact that they can also be prepared using sol-gel methods. However, conventional sol-gel techniques usually require the use of harsh reaction conditions such as high pressures and/or temperatures, extremes in pH and the use or generation of caustic chemicals. On the contrary and in nature, the production of amorphous biosilica can be accomplished under mild conditions. Recently, we discovered a new biomimetic catalyst for TEOS hydrolysis and condensation: L-glutathionereduced (GSH). Because of its acidic character in aqueous solutions, GSH can be employed alone or titrated against cysteamine (Cys, basic catalyst already reported in literature) for the formation of silica-based materials over broad and tunable ranges of pH (including neutral pH). Therefore, these strategies can be used to achieve the most favorable pH conditions that should be employed for the production of silica-based materials and in terms of the desired final biological properties (such as biocompatibility, cell toxicity and biodegradability) and mechanical/morphological properties (such as stability, porosity, nano-rugosity and surface area). To further improve the osteogenic properties of implanted BGs, several bioactive molecules such as specific growth factors and dexamethasone (DXMT) can be incorporated into these materials. The supercritical carbon dioxide (scCO2) deposition method permits to have previously prepared inorganic materials and later deposit into them a desired scCO2-soluble bioactive molecule, considering the envisaged therapeutic application and without interfering with materials manufacture and/or processing methods. Moreover, this can be performed avoiding the use of high temperatures (which may degrade the involved thermo-labile substances) and of organic solvents (which allow the deposition of those hydrophobic substances that cannot be deposited by aqueous solution/suspension soaking methods) [7-13]. In this work, we prepared bioactive glasses (BGs) (based on SiO2, P2O5, Na2O, CaO) and silica-calcium hydroxyapatite (HA) composite materials using a one-pot aqueous sol-gel method at 37 ºC and at atmospheric pressure [2-4] and using the two above referred biomimetic catalysts (GSH and Cys). Other precursors included triethyl phosphate (TEP), calcium nitrate tetrahydrate (CaNT) and sodium nitrate (NaNO3). Additional composite samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic calcium hydroxyapatite particles or by changing the employed amount of water. In addition, some samples were also obtained by adding chitosan as an organic porogenic agent. All synthesized wet materials were dried and submitted to a specific sinterization process (from 60 ºC up to 1000ºC) [2]. Obtained solid materials were later loaded with DXMT by a scCO2 deposition process or by conventional loading from aqueous dexamethasone solutions. Drug loading yields were changed by controlling scCO2 densities (755-920 Kg/m3) at a constant depressurization rate (0.5 MPa/min) and processing time (14 hours). Loading yields and drug release profiles were obtained spectrophotometrically for both drug loading methods All obtained materials were characterized by mercury intrusion porosimetry, nitrogen adsorption, helium pycnometry, simultaneous differential thermal analysis (SDT), scanning electron microscopy (SEM), infra-red spectroscopy (FTIR) and X-ray diffraction. MATERIALS AND METHODS GSH (Sigma-Aldrich, >99.0%) and Cys (Sigma-Aldrich, >98.0%) were used as biomimetic catalysts. The following chemical were used as precursors for the sol-gel 45S5 synthesis: tetraethyl orthosilicate (Aldrich, >99.0%), triethyl phosphate (Aldrich, >99.8%), sodium nitrate (Sigma-Aldrich, >99.0%) and calcium nitrate tetrahydrate (Sigma-Aldrich, >99.0%). Hydroxyapatite (Agoramat) and chitosan (medium molecular weight, Sigma-Aldrich), dexamethasone (Sigma-Aldrich, >98.0%) and carbon dioxide (99.998%, Praxair, Spain) were also used. Bioactive glasses (BG) (based on SiO2, P2O5, Na2O, CaO) and silica-calcium hydroxyapatite (HA) composite materials were prepared using a one-pot aqueous sol-gel method at 37 ºC and at atmospheric pressure. Biomimetic catalysts aqueous solutions were prepared: GSH (0.15M, pH 2.56-2.66) and Cys (0.15M, pH 8.969.20). TEOS, HA, CHIT and other precursors (including TEP, CaNT and NaNO3) were also and the employed amounts are presented in Table 1. The catalyst solution at neutral pH (GSH+Cys, pH 7.03-7.07, 24.3ºC) was prepared by the titration of the previously prepared GSH and Cys solutions until neutral pH was attained. Additional silica-HA composite samples were obtained by replacing TEP, CaNT and NaNO3 by synthetic HA powders. For other samples, CHIT was added (0.4 g, 2% w/v) to the GSH solution and mixtures were stirred overnight until complete CHIT dissolution. The final pH of this solution was pH 3.10-3.15. All reactions were carried for 25 days, for all the employed catalytic systems. After synthesis, obtained materials were centrifuged (5000 rpm, 10 min), the supernatant was removed and samples were freeze-dried at -47 ºC for 72 hours (Snijders Scientific). Then, samples were grinded and submitted to the sinterization process (at 60, 200, 600 and 1000ºC for 72, 40, 5 and 2h, respectively) [2]. Heating rate was 8ºC/min. Obtained solid materials were later loaded with dexamethasone by a scCO2 deposition process. Obtained BG or HA powders (~90 mg) were placed into sealed dialysis bags and then introduced in a high pressure view-cell which was previously loaded with ~34 mg of DXMT. Drug loading yields were changed by controlling scCO2 densities (755-920 Kg/m3), i.e., by performing experiments at 313K/13.6 MPa, and at 313 K/32.0 MPa. Employed depressurization rate was 0.5 MPa/min and processing time was 14 hours. DXMT-loaded BG and HA powders (~10 mg) were placed into sealed dialysis bags and then immersed and stirred at 310 K in 10 mL of MilliQ water. At pre-determined time intervals, an aliquot was removed and analyzed in a UV-Vis spectrophotometer at 242 nm. Table 1 – Synthesized samples and employed chemical amounts. Catalyst solution (ml) BG BG + CHIT BG + water HAs HA + CHIT CHIT (g) TEOS (ml) TEP (ml) Ca(NO3)2.4H2O (g) NaNO3 (g) MilliQ Water (ml) HA (g) 0.40 - 8.95 8.95 8.95 8.95 8.95 0.67 0.67 0.67 - 5.53 5.53 5.53 - 3.73 3.73 3.73 - 10.0 - 2.35 2.35 20.0 20.0 20.0 20.0 20.0 0.40 All obtained materials were chemically and physically characterized. Mercury intrusion porosimetry (Autopore IV, Micromeritics) was performed to determine pore size distribution while real density was determined by helium pycnometry (Accupyc 13330, Micromeritics). Surface area, pore diameter and pore volume were determined nitrogen adsorption (ASAP 2000 V2.04, Micromeritics). Prepared/processed samples were also characterized by scanning electron microscopy (XL30, Phillips), by FTIR (ATR and transmission modes) spectroscopy (4000, Jasco) and by X-ray diffraction (XRD) (X’Pert, Phillips). Simultaneous differential thermal analyses (SDT) were also carried out (Q600, TA Instruments). RESULTS AND DISCUSSION It was observed that obtained BGs presented similar chemical compositions as those obtained with conventional catalysts [2]. In addition, FTIR confirmed the decomposition of the two nitrates during the sinterization thermal treatment (1042, 820 and 744 cm-1 bands, Figure 1A). A crystalline phase assigned to the P-O deformation modes in crystalline phosphate is also detected (622 and 617 cm-1 bands) in sinterized samples obtained from GSH+Cys and from Cys (Figure 1B). On the other hand, the symmetrical Si-O-Si stretching vibration on SiO44of crystalline silicate is detected after sinterization and for samples prepared by using GSH (729 and 697 cm-1 bands). FTIR also confirmed the presence of characteristic silica bands such as hydrogen-bonded water stretching bands (3470-3450 cm-1), adsorbed water molecules deformation vibrations (1653-1634 cm-1) and surface silanol groups and silicon-oxygen covalent bonds vibrations (1200-1000 cm-1). Si-O-Si symmetric stretching vibration (800 cm-1) and the corresponding bending mode (469-466 cm-1) are also present [2]. XRD spectra of prepared BGs (obtained using the different catalytic systems) show that the Na2Ca2Si3O9 crystalline phase could be identified on that BG obtained from GSH (BG-GSH). On samples obtained from Cys and from GSH+Cys mixtures, it was possible to find a different crystalline phase (mainly Na2Ca3Si6O16) as well as a specific SiO2 crystalline cristobalite form (and as already found by FTIR). The Na3PO4 phase could also be identified in the BG-Cys sample (Figure 2). We recently find that different pH conditions and different biomimetic catalysts (GSH, Cys and GSH+Cys mixtures) strongly affected the final morphological and mechanical properties of silica-based materials prepared by the employed aqueous sol-gel methods [13]. This can also be observed in Figure 3 in terms of the obtained surface areas and real densities of prepared BG and HA samples. 4 BG-GSH A Absorbance, u.a. 2 B BG-Cys 3 Absorbance BG-Cys without Thermal Treatment BG-Cys with Thermal Treatment BG-GSH+Cys 1 0 3400 2400 1400 Wavenumber, cm-1 400 3400 2400 1400 400 Wavenumber, cm-1 Figure 1. FTIR spectroscopy of some selected BG samples with/without sinterization thermal treatment (A) and sinterized BGs obtained using different catalysts (B). BG- GSH BG-Cys BG- GSH+Cys 10 20 30 40 50 60 70 80 2θ(o ) Figure 2. XRD patterns of BGs obtained using different catalysts. Higher surface area and lower real density were obtained for BGs prepared by using a GSH+Cys catalytic system (Figure 3A). Despite they present similar real densities, these samples presented much lower surface areas than those prepared with HA (Figure 3B). In fact, the obtained higher surface areas in HA samples is certainly related to the presence of added calcium hydroxyapatite particles, since there is no HA shrinkage during the sinterization process (as happens with its silica counterpart). The employed porogenic agent (CHIT) did not greatly affect the surface areas and real densities of prepared BGs but it dramatically increased the surface area of prepared HA composites. However, the real density of these materials are quite similar to other prepared BG and HA composites. A GSH Cys GSH/Cys GSH + CHIT GSH + H2O B GSH/HA Cys/HA GSH/Cys/HA GSH/HA + CHIT Figure 3. Selected morphological characterization results of prepared BGs and HA samples with different catalytic systems: ■ surface area (m2/g) and □ real density (g/m3). This can also be verified by SEM results (Figure 4). Biomaterials surface areas, pore sizes and geometries, pore volumes and densities, as well as surface rugosities are very important parameters to consider in tissue engineering applications. Despite all prepared samples present quite rugged surfaces, those composites formed by silica and HA clearly present much higher surface rugosities (for all employed catalytic systems). Mesoporous materials (1.5 nm < average pore diameters < 15-57 nm) were obtained for all prepared BG and HA samples (results not presented). As an example, the thermogravimetric and calorimetric results of a BG sample prepared using GSH as the catalyst are shown Figure 5. The thermal events associated to the sinterization process (> 600 ºC), to the removal of water and of un-reacted TEOS (50-180 ºC) and of GSH degradation (200 ºC) can be clearly seen [6, 13]. The sinterization process started around 700 ºC (with nitrate and silanol removal) and continued by crystallization (900-1200 ºC), probably leading to the formation of a cristobalite phase, as previously indicated by XRD results. Cristobalite is a high-temperature silica polymorph that usually forms between 945 °C and 1085 °C [14]. BG-GSH BG-Cys BG-GSH+Cys A BG-GSH/HA BG-Cys/HA BG-GSH+Cys/HA B Figure 4. Selected SEM microscopy results of prepared BGs and HA samples with different catalytic systems. 20 8 15 Weight, mg Catalysts removal 5 0 6 Onset of crystallization and silica polymorphism 5 Nitrate and silanol removal 4 -5 -10 Heat Flow (mW) 10 7 -15 -20 Water and chemical residuals removal -25 3 -30 0 200 400 600 800 1000 1200 Temperature, ºC Figure 5. Thermogravimetric and calorimetric SDT profiles for a BG obtained using GSH as catalyst. DXMT loading was carried out by scCO2 deposition into prepared BG and HA samples. Sustained drug release profiles were always observed for all prepared and processed BG and HA samples. A selected example is shown in Figure 6. Even after the presented 10 h release period, it can be observed that scCO2-loaded DXMT was not completely released from BG samples. It is also clear that samples loaded at lower density conditions can uptake more DXMT. This can be explained by a higher DXMT affinity for BG than for the scCO2 phase and it is a common behavior on the impregnation/deposition of those additives presenting relatively low solubilities in scCO2 and favorable interactions with the material to be loaded. Dexamethasone/BG, µg/mg 0.25 0.20 0.15 0.10 0.05 0.00 0 2 4 6 8 10 Time, h Figure 6. Dexamethasone release from BG samples obtained using Cys as catalyst. Samples were processed for 14h and depressurization rate was 0.5MPa/min. Loading was performed at different scCO2 densities: (○) 755 Kg/m3 (13.6 MPa, 313 K) and (●) 920 Kg/m3 (32.0 MPa, 313 K). CONCLUSIONS Different silica formation pH conditions, different biomimetic catalysts (GSH, Cys and GSH+Cys mixtures) employed at distinct concentrations and the use of chitosan as a porogenic agent, strongly affected the final morphological and mechanical properties of BGs and of silica-HA composite materials prepared by the employed aqueous sol-gel method. Consequently, these different properties also had some effect on other relevant properties for the intended applications such as DXMT loading yields, DXMT release profiles and materials biological properties (not presented). Nevertheless, these results demonstrated the feasibility and the associated advantages of the employed aqueous sol-gel method and of the scCO2 deposition process for the development of dexamethasone-loaded BGs and silica/HA composites for potential biomedical and tissue engineering applications. However, more work still needs to be performed in order to deeply understand the already obtained results as well as to optimize the efficiencies of the employed experimental methodologies This will include the study of other related composite systems and compositions, and the optimization of the scCO2 deposition method operational conditions, of the drug release experimental conditions and methodologies, and of the hemocompatibility/ biological assays. REFERENCES [1] ARCOS D., VALLET-REGÍ M., Acta Biomater, Vol. 6, 2010, p 2874. [2] CHEN Q., LI Y., JIN L., QUINN J.M.W., KOMESAROFF P.A., Acta Biomater, Vol. 6, 2010, p. 4143. [3] RAHAMAN M.N., DAY D.E., BAL B.S., FU Q., JUNG S.B., BONEWALD L.F., TOMSIA A.P., Acta Biomater, Vol.7, 2011, p.2355. [4] VALLET-REGÍ M. J Chem Soc. Dalton Trans, 2001, p.97 [5] RAGEL C.V., VALLET-REGÍ M., RODRÍGUEZ-LORENZO L.M., Biomaterials, Vol.23, 2002, p.1865. [6] JONES J.R., EHRENFRIED L.M., HENCH L.L., Biomaterials, Vol. 27, 2006, p.964. [7] KIKIC I., VECCHIONE F., Curr Opin Solid St Mat Sci, 2003, 7, 399-405. [8] KAZARIAN S.G., Polym Sci Series C, Vol.42, 2000, p.78. [9] BRAGA M.E.M., PATO M.T.V., COSTA SILVA H.S.R., FERREIRA E.I., GIL M.H., DUARTE C.M.M., SOUSA H.C., J Supercrit Fluids, Vol. 44, 2008, p.245. [10] DIAS A.M.A., BRAGA M.E.M., SEABRA I.J., FERREIRA P., GIL M.H., SOUSA, H.C. Int J Pharm, Vol. 408, 2011, p.9. [11] COSTA V.P., BRAGA M.E.M., GUERRA J.P., DUARTE A.R.C., DUARTE C.M.M., LEITE E.O.B., GIL M.H., DE SOUSA H.C., J Supercrit Fluids, Vol 52, 2010, p. 306. [12] COSTA V.P., BRAGA M.E.M., DUARTE C.M.M., ALVAREZ-LORENZO C., CONCHEIRO A., GIL M.H., DE SOUSA H.C., J Supercrit Fluids, Vol 53, 2010, p. 165. [13] BRAGA M.E.M., CHIM R.B., ALVES M.C., ZIEGLER C.R., WATKINS J.J., DE SOUSA H.C. In: Proceedings of the 13th EMSF, European Meeting on Supercritical Fluids, Den Hague, The Netherlands, October 9-13, 2011. ISBN 978-2-905267-77-1. [14] VERDUCH A. G., J Am Ceramic Soc Vol 41, 1958, p. 427.
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