Synthesis of nanosized hydroxyapatite/agarose ...

Materials Today Communications 8 (2016) 31–40

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Synthesis of nanosized hydroxyapatite/agarose powders for bone filler and drug delivery application Elayaraja Kolanthai a,b , Kathirvel Ganesan c , Matthias Epple c , S. Narayana Kalkura a,∗ a

Crystal Growth Centre, Anna University, Chennai 600 025, Tamil Nadu, India Central Research Laboratory, Sree Balaji Medical College & Hospital (SBMCH), Bharath University, BIHER, Chrompet, Chennai 600 044, India Institute of Inorganic Chemistry and Center for Nanointegration Duisburg-Essen (CeNIDE), University of Duisburg-Essen, Universitaetsstrasse 5-7, 45117 Essen, Germany b c

a r t i c l e

i n f o

Article history: Received 25 February 2016 Received in revised form 12 March 2016 Accepted 17 March 2016 Available online 16 May 2016 Keywords: Hydroxyapatite Agarose Composites Mesoporous Amoxicillin 5-Fluorouracil drug delivery

a b s t r a c t Drug-loaded bioactive composite powders are used for the treatment of orthopedic diseases and prevention of infection or inflammatory reaction after surgical implantation. Nanosized (80 × 23 nm2 ) and porous (17 ± 1 nm) hydroxyapatite (HAp)/agarose composite rods were prepared by sol-gel synthesis and subjected to microwave and conventional heating. Microwave heating increased the degree of crystallinity and the thermal stability and produced calcium-deficient HAp/agarose composite powders. There was a considerable reduction (by 39%) in the size of rods on microwave heating whereas the conventional heating at 700 ◦ C rendered the samples porous and agglomerated with a significant decrease in the specific surface area. The agarose contents in as-synthesized and microwave heated samples were ∼14% and 4%, respectively. The samples were partially degradable upon immersion in SBF, and later exhibited calcium phosphate deposition which was confirmed by gravimetry. An antibiotic (amoxicillin) and anticancer (5-fluorouracil) drug-loaded microwave-heated nanosized HAp/agarose composite powder gave an extended drug release when compared to the as-synthesized and the conventionally heated samples. The composite powders showed a negative zeta potential, hemocompatibility and better antimicrobial efficacy than pure HAp (conventional heated sample). The microwave heating retained the organic phase (agarose) along with a reduction in particle size. In addition, this technique is simple, fast and costeffective to produce mesoporous, bioactive and resorbable nanocomposite (HAp/agarose) powders which could find application as bone filling materials and drug delivery systems. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Nowadays, there is an increasing incidence of bone-related diseases such as osteomyelitis (infection) and cancer [1]. Osteomyelitis is an inflammation of bone or bone marrow, usually caused by bacteria or fungi. These microorganisms infect and spread to the bones and adjacent areas through the blood stream [2]. The treatment of these diseases consists of chemotherapy, tissue transfers, bone grafting and the implantation of antibioticloaded biocompatible materials (beads, paste or solids). Infection is a major problem in the medical field due to poor accessibility of the infected site by systemically administered antibiotics [3]. Hence, an efficient and controlled local drug delivery system has to be developed, and current research efforts are directed to develop novel

∗ Corresponding author. E-mail address: [email protected] (S.N. Kalkura). http://dx.doi.org/10.1016/j.mtcomm.2016.03.008 2352-4928/© 2016 Elsevier Ltd. All rights reserved.

drug storage and release systems [3]. The use of nanobiomaterials such as biodegradable polymers [4], xerogels [5], hydrogels [6], mesoporous silica [7], calcium phosphate (hydroxyapatite) [8] and polymer composites [9] as drug carriers leads to greater efficiency, safety, biocompatibility and provides a better therapeutic response due to controlled and prolonged release of the drugs [4]. Hydroxyapatite, Ca10 (PO4 )6 (OH)2 , HAp, is the major inorganic component of bone and teeth. Nanosized HAp has a considerable potential to be used as implant, prosthetic bone replacement and as protein and drug delivery system [8,10]. Heated HAp has a lower bioactivity and is brittle in nature; hence it is not suitable for load bearing applications. The powder form of HAp is good for filling bone cracks and small irregular defects. However, there will be a migration of HAp particles from the implants. It is difficult to handle and keep the implant compact in the defect site [11,12]. Thus, it is necessary to mix a suitable biocompatible polymer with the HAp granules to overcome these drawbacks. HAp composites have attracted much attention as the presence of HAp in the

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E. Kolanthai et al. / Materials Today Communications 8 (2016) 31–40

composite material enhances the proliferation of osteoblasts, resulting in a better osteoconductivity [13,14]. Composites based on degradable biopolymers such as collagen [15], fibrin glue, gelatin, chitosan, alginate and hydroxy-propyl-methyl cellulose with inorganic powders were reported as bone fillers [16]. Agarose is a natural, thermally responsive polysaccharide that is widely used in biological sciences (e.g., microbial cultivation and gel electrophoresis) [17]. It is a linear polymer behaving like a hydrogel, allowing rapid room temperature polymerization [18,19]. Studies have demonstrated the suitability of agarose scaffolds for promoting stem cell differentiation into chondrocytes [20]. Tabata et al. [21], and Suzawa et al. [22] reported that HAp/agarose and calcium carbonate/agarose scaffolds had better healing properties than pure HAp. The semi-synthetic orally administered broad-spectrum antibiotic drug amoxicillin (AMX) is extensively used against bacterial infections. A slow and continuous release of the drug during bone implantation is essential to prevent infectious diseases. A slow and continuous release from the drug-loaded calcium phosphates, porous HAp blocks and HAp coated on metals was previously reported [23–25]. 5-Fluorouracil (5-Fcil) is an antineoplastic drug which is used in the treatment of cancer. It is an acidic, water-soluble and hydrophilic drug used in chemotherapy for the treatment of solid tumors [26,27]. To the best of our knowledge, there are no reports on the synthesis of nanocrystalline HAp/agarose composite powders by microwave heating. Here, we report the synthesis of nanosized HAp/agarose composite by a pHcontrolled sol-gel technique. In addition, the effects of microwave and conventional heating on the biological and drug release properties were investigated. 2. Experimental methods 2.1. Material preparation The powder form of nanosized HAp/agarose composite was prepared by an ethanol-based sol-gel technique, followed by microwave treatment. Calcium nitrate tetrahydrate Ca(NO3 )2 ·4H2 O (Merck), diammonium hydrogen phosphate (NH4 )2 HPO4 (Merck), agarose (SRL), ethanol (Merck) and aqueous ammonia solution (Merck) were used for the synthesis without further purification. 0.3 M diammonium hydrogen phosphate (9.91 g) was dissolved in 250 mL of ethanol, heated to 85 ± 5 ◦ C and subjected to vigorous stirring. Then, 1 wt% of agarose was added to the phosphate solution. 0.5 M calcium nitrate tetrahydrate

(29.51 g) was dissolved in 250 mL ethanol which was added to phosphate/agarose solution at constant flow rate (2 mL/min) under continuous stirring. After mixing, the solution was continuously stirred for 3 h. The pH of the solution was maintained at 10.5 by the addition of aqueous ammonia solution with a pH stat instrument (Radiometer analytical). A constant temperature of 85 ± 5 ◦ C was maintained until the end of the reaction. The reaction mixture was refluxed until the completion of the reaction to avoid the evaporation of the solvent ethanol/water. The solution was vigorously stirred, aged for a day and final precipitates washed with deionized water. The colloidal precipitates were centrifuged at 3000 rpm and dried at 70 ◦ C in air. The same procedure was applied to prepare colloidal precipitates and it was subjected to microwave heating at 900 W for 30 min (Whirlpool model magicook). The as-synthesized powder was subjected to conventional heating at 700 ◦ C in air for 2 h. After heating, a color change was observed on the powders (brown to white) due to the decomposition of agarose. The schematic illustration for preparation of HAp/agarose composite by sol-gel synthesis and subjected to heating by microwave and conventional is shown in Fig. 1. The as-synthesized samples, microwave and conventionally heated samples are referred to as SAS, SMWT and SAS700, respectively in the following. 2.2. Characterization Powder X-ray diffraction patterns were recorded with a Siemens D500 diffractometer (CuK␣ radiation, = 1.5406 Å, 40 kV, 20 mA, 5–70 degree 2, increment steps of 0.02 degree 2). The diffraction peaks were indexed and its full width half maximum was analyzed with the XRDA software [28]. Further, the crystallinity was examined by empirical relation between Xc and ␤z i.e. ␤z × (Xc )1/3 = KA with Xc the degree of crystallinity, ␤z the full width at half maximum of (002) plane in (◦ 2), and KA a constant set to 0.24 [29]. Fourier transform infrared (FTIR) spectra were recorded in the range of 400–4000 cm−1 with a Perkin-Elmer spectrometer RXI FTIR with KBr pellets. The surface morphology and the elements present in the samples were studied by scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDX) ESEM Quanta 400 FEG, FEI; gold-palladium [80:20]sputtered samples; EDX detector: S-UTW-Si (Li) [30]. Transmission electron microscopy TEM was performed using a JEOL 2100 microscopy operating at 200 kV with 0.1 nm point resolution and equipped with a Gatan US1000, 2048 × 2048 pixel CCD camera. 10 mg of synthesized samples were dispersed in 5 mL ethanol using probe type sonicator for 5 min and deposited on an Augar

Fig. 1. Schematic illustration for synthesis of nanosized HAp/agarose powder by sol-gel synthesis and subjected to heating by microwave and conventional.


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Scientific 300 ␮m holey carbon grid which was allowed to dry in air. The specific surface area was determined by the BrunauerEmmett-Teller (BET) method using a Micromeritics (model-ASAP 2020 V3.00 H) surface area analyzer. The pore size and volume were obtained from the BJH absorption/desorption isotherm. The thermogravimetric analyses (TGA, Perkin Elmer Diamond TG/DTA) of SAS, SMWT and SAS700 were carried out from 30 to 900 ◦ C under nitrogen atmosphere at a heating rate of 20 K/min. The particle size was measured by dynamic light scattering (DLS) with a Zetasizer Nano-ZS (Malvern, UK). A He-Ne diode laser light of wavelength 633 nm as the source was scattered at a fixed angle of 90◦ at room temperature. Powdered samples of 1 mg in 10 mL were dispersed in deionized water, followed by ultra-sonication for 20 min. All measurements were performed in triplicate. 2.3. In vitro dissolution study Phosphate buffer saline (PBS) was prepared by dissolving the chemicals in deionized water by the following order: 8 g NaCl, 0.2 g KCl, 0.2 g KH2 PO4 , 1.15 g NH2 HPO4 for 1 L. It’s the pH was adjusted to 7.4 with HCl. The samples SAS, SMWT and SAS700 were processed into pellets with 8 mm diameter and 1 mm thickness with a uniaxial hydraulic press. These pellets were immersed in 20 mL of PBS in plastic containers and incubated at 37 ◦ C for different time intervals (one to four weeks). After dissolution study, the samples were dried at 80 ◦ C for 3 days under vacuum condition. Finally, the weight difference between before and after soaked samples were measured after four weeks (W) with a Sartorius balance with an accuracy of ±0.01 mg. The pH of the dissolution medium was measured after removing the samples (1 week, 2 weeks, 3 weeks and 4 weeks) from the PBS solution.

Fig. 2. XRD patterns of (a) SAS (b) SMWT and (c) SAS700.

2.4. In vitro bioactivity study In vitro bioactivity of SAS, SMWT and SAS700 were investigated to form bone like apatite on the surface using simulated body fluid (SBF). It was prepared using reagent grade chemicals dissolved in the following order: NaCl, NaHCO3 , KCl, Na2 HPO4 ·2H2 O, MgCl2 ·6H2 O, CaCl2 ·2H2 O, Na2 SO4 , TRIS buffer and 1 M HCl in deionized water. Finally, HCl was used to adjust pH 7.4 at 37 ◦ C [31]. All samples were pressed into pellets (8 mm diameter and 1 mm thickness) with constant weight (100 mg) and was immersed into 20 mL of SBF in polyethylene containers at 37 ◦ C for one to four weeks. The solution was exchanged every two days. After incubation, the samples were gently washed with deionized water and dried at 37 ◦ C for analysis. The samples were weighed with an accuracy of 0.01 mg before and after soaking. Apatite deposition on the surface of the pellet was investigated by SEM. 2.5. Hemolysis test Fresh human blood (author KE blood) was collected in a sterile centrifuge tube which contained heparin to avoid the clot formation. 100 mg of SAS, SMWT and SAS700 of pellets were equilibrated by 1 mL of sterile saline and incubated at 37 ◦ C for 12 h. After incubation, the saline solution was removed. Then, 250 ␮L of human blood was added on the pellets and incubated for 20 min. Finally, 5 mL of sterile saline was added on each sample to stop the hemolysis and were incubated for 1 h [27]. The positive and negative controls were obtained by adding 250 ␮L of human blood to 4.75 mL sterile distilled water and 4.75 mL sterile saline and incubation at 37 ◦ C for 1 h. All samples were centrifuged at 1000 rpm for 5 min. The absorbance (optical density) of the supernatant solution was recorded at 545 nm with a UV–vis spectrophotometer (Shimadzu, UV-1601). The percentage of hemolysis was calculated with the following formula [32]:

Fig. 3. FTIR spectrum of (a) SAS (b) SMWT and (c) SAS700.

Percentage of hemolysis = ([OD (test) − OD (negative control)]/[OD (positive control) − OD (negative control)]) × 100 The accepted norm of hemolysis the percentage is (i) highly hemocompatible (20% hemolysis) [32]. 2.6. Drug loading/release The AMX absorption maximum (max ) was at 230 nm (UV–vis spectrophotometry). A standard graph was obtained by plotting concentration versus absorbance. A best linear fit with a correlation coefficient of 0.9998 was taken for the drug release calculation. For, 5-Fcil maximum absorption wavelength (␭max ) was 265 nm and the standard graph regression value was 0.9994. In vitro drug release experiment was carried out with antibiotic(Amoxicillin, AMX) and anticancer- (5-Fluorouracil, 5-Fcil) loaded HAp pellets. The SAS, SMWT and SAS700 powder samples were mixed with AMX and 5-Fcil powder in the ratio 1:0.5 (samples

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Table 1 Lattice parameters and unit cell volume of SAS, SAS700 and SMWT. Samples code

Lattice parameters

SAS

a and b = 9.44 ± 0.04 Å c = 6.87 ± 0.01 Å V = 530.69 Å3 a and b = 9.42 ± 0.02 Å c = 6.87 ± 0.01 Å V = 528.61 Å3 a and b = 9.30 ± 0.02 Å c = 6.86 ± 0.01 Å V = 513.55 Å3

SMWT

SAS700

100 mg and drug 50 mg, respectively) and made to 8 mm pellet using a hydraulic press with constant pressure (2 t). The drugloaded pellets were immersed in 200 mL of PBS with pH 7.4, and kept in a conical flask with shaking speed of 100 rpm maintained at 37 ◦ C in an orbital shaker (Niolab). From the dissolution medium, 1 mL of AMX and 5-Fcil solutions were removed at various time intervals and replaced by the same volume of PBS. The quantity of drug release was determined by UV spectroscopy (␭max = 230 nm for AMX and 265 nm for 5-Fcil). All samples were prepared in triplicate and the average value of the data was used [23,24].

2.7. Antimicrobial activity test SAS, SMWT and SAS700 powder was mixed with AMX powder in the ratio 1:0.5 (Samples 100 mg and drug 50 mg, respectively) and made to 8 mm pellet using hydraulic press with constant pressure (2 t). The bactericidal effect of SAS, SMWT and SAS700 with AMX drug-loaded pellets was investigated with the Gram-negative bacterium Escherichia coli (E. coli, MTCC 2939) and the Gram-positive bacteria Staphylococcus aureus (S. aureus, MTCC 3381), Staphylococcus epidermidis (S. epidermidis, MTCC 3382). A qualitative diffusion disk test was carried out using 105 colony forming units (CFU/mL) of E. coli, S. aureus and S. epidermidis. The culture was added to the plate and was spread uniformly on Muller-Hinton agar plates. After spreading, 8 mm pellets of the samples were placed onto the culture plate. The plates were incubated at 37 ◦ C for 24 h. The inhibition zone was measured and the photographically documented [23,24].

3. Results and discussion 3.1. XRD analysis The XRD patterns of SAS, SMWT and SAS700 are shown in Fig. 2(a–c). The patterns were in good agreement with the JCPDS

Fig. 4. TGA of (a) SAS (b) SMWT and (c) SAS700.


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Fig. 6. DLS size distribution plot of (a) SAS (b) SMWT and (c) SAS700. Fig. 5. SEM-EDX of (a) SAS (b) SMWT, (c) SAS700 and TEM-SAED image of (d) SAS, (e) SMWT and (f) SAS700.

data for hydroxyapatite (09-0432). The SAS showed broad peaks of low intensity, indicating the presence of nanosized particles and agarose (Fig. 2a). There was an increase in the intensities of (211), (112), (100), (101), (200) and (111) peaks of SMWT (Fig. 2b) whereas for SAS700, there was an overall increase in peak intensities combined with a significant reduction in the peak width (FWHM) (Fig. 2c). This may be due to the decomposition of agarose from SAS700, during the conventional heating process. The increased peak intensity along with a decrease in FWHM of the diffraction patterns indicated an increase in the crystallinity of

SMWT and SAS700. Compared to the SAS700, SMWT had retained the organic phase. During microwave treatment, there is a uniform heating in the whole matrix, leading to a higher crystallinity and a reduction in the crystallite size. Lattice parameters and unit cell volume decreased significantly in both type of heating (Table 1). 3.2. FTIR analysis FTIR spectra of SAS, SMWT and SAS700 are shown in Fig. 3(a–c). The intense broad band between 2600 and 3750 cm−1 of SAS was ascribed to the OH stretch of hydroxyl groups along with the NH stretch of NH4 + . The sharp peak at 1386 cm−1 was assigned to

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the C O C stretch of the agarose [33,34]. The bending mode of water was observed at 1638 cm−1 , and the band at 1034 cm−1 was assigned to the triply degenerate asymmetric P O stretching mode (3 ). The low intensity peak at 962 cm−1 was due to the non-degenerate P O symmetric stretching mode (1 ). The band at 825 cm−1 was assigned to NO3 − bending mode. The peak at 637 cm−1 was attributed to the hydroxyl stretch vibration. The well resolved peaks at 606 and 562 cm−1 were ascribed to the triply degenerate O P O bending mode (4 ). The doubly degenerate of O P O bending mode was observed at 475 cm−1 . SAS spectrum provides the evidence for the formation of HAp/agarose composite (Fig. 3a). A low intensity peak at 1383 cm−1 was assigned to C O C stretch of the agarose which confirmed the presence of agarose even after microwave heating (Fig. 3b). The peak at 1383 cm−1 was of low intensity compared to SAS, may be due to the presence of small quantity of agarose even after the microwave heating. For SAS700, there was a decrease in intensity between 2600 and 3750 cm−1 (Fig. 3c). The reduction of peak intensity at 1638 and 3467 cm−1 indicates the loss of water and NH4 + respectively. The sharp peak observed at 3570 cm−1 was attributed to hydroxyl stretch in HAp. In addition, the phosphate peaks at 1034 and 637 cm−1 were well defined revealing the improved crystallinity and phase purity of HAp [35]. The absence of a peak at 1386 cm−1 , indicates the complete decomposition of agarose after heating. The appearance of low intensity peaks at 1460 and 1415 cm−1 were attributed to the carbonate group vibration due to the agarose decomposition and chemisorption of atmospheric CO2 during the conventional heating [36]. 3.3. TGA Thermogravimetric curves of the samples are shown in Fig. 4(a–c). SAS and SMWT, respectively, showed 6% and 1% mass loss below 200 ◦ C, corresponding to the loss of water (Fig. 4a and b). Weight losses of 13% and 4%, respectively, occurred for SAS and SMWT between 200 ◦ C and 450 ◦ C due to the pyrolysis of agarose [37,38]. The weight loss of around 5% and 1% between 500 and 900 ◦ C was attributed to the condensation of the hydrogenphosphate group (HPO4 2− ) of HAp. In the case of SAS700, first weight loss (1%) occurred below 320 ◦ C due to the loss of adsorbed water. The second weight loss (2%) between 500 ◦ C and 900 ◦ C was

Fig. 7. Nitrogen adsorption/desorption isotherms of (a) SAS (b) SMWT and (c) SAS700.

due to the decomposition of carbonate and condensation of the hydrogen-phosphate group of HAp (Fig. 4c). SAS700 showed an enhanced thermal stability compared to SAS and SMWT, due to the pure phase of HAp. The agarose contents of SAS and SMWT were 13% and 4%, respectively. 3.4. Surface analysis SEM micrographs and EDX spectra of SAS, SMWT and SAS700 are shown in Fig. 5(a–c). Rods of HAp/agarose were observed in SAS and SMWT (Fig. 5a–b). SAS700 showed agglomerated particles with non-uniform interconnected pores (100–150 nm) formed due to the decomposition of agarose (Fig. 5c) [39]. EDX gave the Ca/P ratio of SAS, SMWT and SAS700 to be 1.66, 1.54 and 1.68, respectively. Composition and structure of calcium-deficient HAp/agarose composites are almost similar to that of HAp [40]. However, it had a higher thermal stability and solubility compared to pure HAp. The advantage of calcium deficient hydroxyapatite over other calcium phosphates include superior seeding efficacy, higher specific surface area and high efficiency of precipitation of bonelike apatites, which has made calcium deficient hydroxyapatite a potential candidate in the area of bone substitution applications [40]. TEM-SAED images of SAS, SMWT and SAS700 are shown in Fig. 5(d–f). The rods of HAp/agarose composite of average size, 80 × 23 nm was observed in SAS (Fig. 5d) whereas, the average size of the rods (49 × 30 nm2 ) was found to be reduced on microwave heating (Fig. 5e). The respective aspect ratio of rods (length/width) was 3.5 ± 0.5 and 1.6 ± 0.3 for SAS and SMWT. SAS700 had agglomerated particles with average size of 65 × 58 nm2 (Fig. 5f). The selected area electron diffraction (SAED) pattern of all the samples showed concentric ring patterns with spots indexed to the (002), (211), (310), (222) and (213) planes of hexagonal phase of HAp (Fig. 5d–f). In addition, concentric ring patterns with spots were observed, indicating the nanosized polycrystalline nature of the samples [8]. 3.5. Particle size and zeta potential analysis Particle size distributions (PSD) and zeta potential of SAS, SMWT and SAS700 are shown in Fig. 6(a–c) and Table 2. The average hydrodynamic particle size, polydispersity index (PDI) and zeta potential of SAS were 300 nm, 0.583 and −18 mV, respectively (Fig. 6a), revealing the polydispersion of the particles with negative zeta potential. The microwave heating reduced the average particle size

Fig. 8. Weight difference of before and after SBF soaked SAS, SMWT and SAS700 (W represents week).


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Fig. 9. SEM micrograph of (a) SAS (b) after SBF soaked SAS (c) SMWT (d) after SBF soaked SMWT (e) SAS700 and (f) after SBF soaked SAS700 samples.

Table 2 DLS-Zeta potential values. Samples

SAS SMWT SAS700

Table 3 Pore size, volume and surface area for SAS, SAS700 and SMWT samples.

Average hydrodynamic particle diameter (nm)

Polydispersity index (PDI)

300 190 800

0.583 0.324 0.455

Zeta Potential (±1 mV)

−18 −16 −15

Sample code

Pore size (Dp ) (nm)

BET Surface area (SBET ) (m2 /g)

Pore volume (Vp) (cm3 /g)

SAS SMWT SAS700

17 24 25

47.8 ± 0.2 41.7 ± 0.1 25.0 ± 0.1

0.3229 0.4502 0.2771

3.6. BET analysis

to 195 nm (Fig. 6b). Conventional heating considerably increased the particle size (800 nm) and zeta potential (7%) (Fig. 6c). The negative zeta potential of the particle did not show significant decrease upon removal of the agarose by the heating process (Table 2). The surface properties such as roughness, chemical composition, surface potential and porosity may help in the binding of cells to these biomaterials [41]. The surface potential analysis of bone implants is of great interest, because it plays an important role in initiating the new bone formation. The negatively charged surface of the bone substitutes may assist the osteoblast cell adhesion[42].

The nitrogen adsorption/desorption isotherms of SAS, SMWT and SAS700 are shown in Fig. 7(a–c). The samples showed type IV isotherms and the typical H1 -hysteresis loops, demonstrating the properties of a typical mesoporous material [43]. The BET surface area, pore volume, pore size of the samples are presented in Table 3. SAS and SMWT had 52% more surface area due to the presence of nanosized particles compared to SAS700. The variation in the surface area of SAS, SMWT and SAS700 samples was also reflected in their pore volume. SAS had a lower pore size than other samples, due to the presence of higher amount of agarose which was further confirmed by the high intensity IR band of agarose (1386 cm−1 ) (Fig. 3a) and higher weight loss in TGA (Fig. 4a). The mesoporous

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Fig. 10. Weight loss graph of SAS, SMWT and SAS700 during the dissolution test.

Fig. 12. In vitro drug release of (a) AMX and (b) 5-Fcil drug (Insert SMWT sample drugs release profile). Fig. 11. Percentage of hemolysis for SAS, SMWT and SAS700.

materials with homogeneous pores, high pore volume and surface area, helps drug-loading and release kinetics [44]. The presence of mesopores (∼50 nm) in the HAp/agarose composite (SAS and SMWT) could enhance the drug delivering properties. 3.7. In vitro bioactivity study The in vitro bioactivity of the sample was tested using simulated body fluid for a period of one to four weeks [31]. SBF-soaked SAS and SMWT showed 8 and 10% weight loss, respectively, during the first week. The weight loss may be due to the dissolution of agarose from the samples in SBF solution, whereas no weight loss was observed in SAS700. All samples showed a gradual increase of weight after one week of immersion in SBF, suggesting the formation of the apatite layer (Fig. 8). The SAS and SMWT showed initial resorbability followed by bioactivity. Micrographs of all samples before soaking in SBF solution showed a non-uniform rough surface (Fig. 9(a, c and e)). Spherical apatite-containing micro pores (1–5 ␮m) were visible on the surface (Fig. 9b, d and f) of the samples soaked in SBF for two weeks. Apatite deposition increased with an increase in soaking time, leading to the formation of a dense layer. SMWT showed an enhanced apatite formation compared to SAS (Fig. 8).

3.8. In vitro dissolution study The in vitro dissolution of the samples was measured in PBS. The experiment was carried out in triplicate for each sample. The pH of the PBS decreased from 7.4 to 7.1 for SAS and SMWT when measured periodically for four weeks, indicating a partial dissolution of the pellet. The SAS showed a higher dissolution rate compared to other samples due to the presence of agarose (Fig. 10). The weight loss of SMWT was 40% less than SAS, due to the comparatively low quantity of agarose (4%) present in SMWT. There was no significant weight loss of SAS700 due to the presence of a stable phase of pure HAp. 3.9. Hemolysis test The percentage of hemolysis was less than one for all the samples (Fig. 11), suggesting that all samples were highly hemocompatible, therefore they may be used for bone filling, wound-dressing or as local drug delivery system [32]. 3.10. In vitro drug release The cumulative percentage of AMX (antibiotic) release profiles for the various samples as a function of release time in PBS are shown in Fig. 12a. SAS showed an initial burst release of about 60%


within 10 h, due to the dissolution of agarose in PBS. Subsequently controlled drug release was observed. The sustained release was attributed to a strong interaction between the mesoporous surface of HAp and the drug. SMWT released 20% of drug in 1 h and afterwards, it displayed sustained release. Each hour, SWNT released 2–3% of drug, and the controlled and extended drug release was observed up to 125 h (100%). Compared with SAS, SMWT had a smaller surface area and a larger pore size. However, SMWT showed a controlled and extended drug release due to its high crystallinity with reduced particle size [45]. SAS700 showed a 100% rapid release within 65 h due to high porosity (Fig. 12a). The cumulative percentage of drug release profiles for the 5-Fcil (anticancer) drug-loaded SAS, SMWT and SAS700 (Fig. 12b) showed a rapid release, followed by a more continuous release. A gradual increase in the percentage of drug release up to 95 h on SMWT was observed. The SAS and SMWT displayed a significant decrease in 5Fcil release, indicating a stronger bonding of 5-Fcil to the samples due to the presence of agarose. In addition, a low pore size and high surface area would also have contributed to the decrease in drug release. A prolonged 5-Fcil drug release would efficiently kill cancer cells during chemotherapy.

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Table 4 Inhibition zone around of drug–loaded samples against E. coli, S. epidermidis and S. aureus. Samples

SAS SMWT SAS700

Diameter of zone of inhibition (±1 mm) E.coli

S. aureus

S. epidermidis

55 55 54

42 44 41

55 49 53

3.11. Antimicrobial activity AMX loaded samples gave an excellent inhibitory effect against S. aureus, S. epidermidis and E. coli (Table 4). From these results, we conclude that AMX drug possess a resistance against the strains S. aureus, S. epidermidis and E. coli. The efficiency against E.coli showed the largest highest zone of inhibition in all samples compared with S. aureus and S. epidermidis (Fig. 13). 4. Conclusion HAp nanorod/agarose composite powders (80 × 23 nm2 ) were synthesized by sol-gel technique at low temperature and constant

Fig. 13. Inhibition zone of without and with AMX loaded (a) SAS (b) SMWT and (c) SAS700 against E. coli, S. aureus and S. epidermis.

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pH (10.5). The content of agarose in as-synthesized and microwave heated samples was ∼14% and 4%, respectively. Microwave heating increased the crystallinity and thermal stability with a decrease in particle size and zeta potential compared to the as-synthesized sample. Conventionally heated samples at 700 ◦ C did not contain any agarose and exhibited interconnected porosity which could assist osseointegration, local drug delivery and circulation of physiological fluid leading to the formation of new bone. The specific surface area of the as-synthesized and microwave heated samples was higher than that of the conventional heated samples. SMWT showed prolonged antibiotic release up to 125 h, which was two times higher than that of SAS. The as-synthesized and microwave heated samples displayed partial resorbability and excellent bioactivity. The samples were hemocompatible and drugloaded powders were strongly active against the most common bacterial strains. Composite powder could be used for bone filling, drug delivery and for reconstructive surgery applications. Microwave heating leads to the rapid production of homogeneous nanosized particles without affecting the organic phase present in it. Acknowledgments One of the authors (K. E) acknowledges CSIR, India for the award of Senior Research Fellowship (File No: 09/468(0413)/2009-EMRI). The authors thank the DST, India for financial support (Project No. SR/SO/HS-05/2005). References [1] E.V. Giger, B. Castagner, J.-C. Leroux, Biomedical applications of bisphosphonates, J. Control Release 167 (2013) 175–188. [2] C. Pineda, A. Vargas, A.V. Rodriguez, Imaging of osteomyelitis: current concepts, Infect. Dis. Clin. North Am. 20 (2006) 789–825. [3] J. Hendriks, J. Van Horn, H. Van Der Mei, H. Busscher, Backgrounds of antibiotic-loaded bone cement and prosthesis-related infection, Biomaterials 25 (2004) 545–556. [4] L. Di Silvio, W. Bonfield, Biodegradable drug delivery system for the treatment of bone infection and repair, J. Mater. Sci. Mater Med. 10 (1999) 653–658. [5] H.-H. Yang, Q.-Z. Zhu, H.-Y. Qu, X.-L. Chen, M.-T. Ding, J.-G. Xu, Flow injection fluorescence immunoassay for gentamicin using sol-gel-derived mesoporous biomaterial, Anal. Biochem. 308 (2002) 71–76. [6] C.-C. Lin, A.T. Metters, Hydrogels in controlled release formulations: network design and mathematical modeling, Adv. Drug Deliv. Rev. 58 (2006) 1379–1408. [7] M. Manzano, M. Vallet-Regi, New developments in ordered mesoporous materials for drug delivery, J. Mater. Chem. 20 (2010) 5593–5604. [8] R. Vani, S.B. Raja, T. Sridevi, K. Savithri, S.N. Devaraj, E. Girija, et al., Surfactant free rapid synthesis of hydroxyapatite nanorods by a microwave irradiation method for the treatment of bone infection, Nanotechnology 22 (2011) 285701. [9] J. Schnieders, U. Gbureck, R. Thull, T. Kissel, Controlled release of gentamicin from calcium phosphate—poly(lactic acid-co-glycolic acid) composite bone cement, Biomaterials 27 (2006) 4239–4249. [10] M.B. Nair, S.S. Babu, H. Varma, A. John, A triphasic ceramic-coated porous hydroxyapatite for tissue engineering application, Acta Biomater. 4 (2008) 173–181. [11] C. Peniche, Y. Solis, N. Davidenko, R. Garcia, Chitosan/hydroxyapatite-based composites, Biotecnol. Apl. 27 (2010) 202–210. [12] R.E. Holmes, H.K. Hagler, Porous hydroxylapatite as a bone graft substitute in mandibular contour augmentation: a histometric study, J. Oral Maxillofac. Surg. 45 (1987) 421–429. [13] S.-S. Kim, K.-M. Ahn, M.S. Park, J.-H. Lee, C.Y. Choi, B.-S. Kim, A poly(lactide-co-glycolide)/hydroxyapatite composite scaffold with enhanced osteoconductivity, J. Biomed. Mater. Res. A 80A (2007) 206–215. [14] C. Verheyen, J. De Wijn, C. Van Blitterswijk, K. De Groot, P. Rozing, Hydroxylapatite/poly (l-lactide) composites: an animal study on push-out strengths and interface histology, J. Biomed. Mater. Res. 27 (1993) 433–444. [15] F.Y. Hsu, S.-C. Chueh, Y.J. Wang, Microspheres of hydroxyapatite/reconstituted collagen as supports for osteoblast cell growth, Biomaterials 20 (1999) 1931–1936. [16] M. Sivakumar, K.P. Rao, Preparation, characterization, and in vitro release of gentamicin from coralline hydroxyapatite-alginate composite microspheres, J. Biomed. Mater. Res. A 65 (2003) 222–228.

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