Adsorption of gum Arabic on bioceramic nanoparticles

Adsorption of gum Arabic on bioceramic nanoparticles

Available online at www.sciencedirect.com Materials Science and Engineering C 28 (2008) 443 – 447 www.elsevier.com/locate/msec Adsorption of gum Ara...

180KB Sizes 0 Downloads 98 Views

Available online at www.sciencedirect.com

Materials Science and Engineering C 28 (2008) 443 – 447 www.elsevier.com/locate/msec

Adsorption of gum Arabic on bioceramic nanoparticles A.C.A. Roque 1 , O.C. Wilson Jr. ⁎ The Catholic University of America, Department of Biomedical Engineering, BONE/CRAB Lab, 117 Pangborn Hall, 620 Michigan Ave NE, Washington DC 20064, United States Available online 11 April 2007

Abstract Surface modification agents can be used to tailor the surface chemistry and biological activity of bioceramic nanoparticles in very intriguing ways. However, the specific modes of interactions between macromolecules and nanoparticles can be difficult to characterize. The aim of this study was to investigate the adsorption of gum Arabic on hydroxyapatite (HAp) and magnetic nanoparticles (MNP) using the bicinchoninic acid (BCA) test. Gum Arabic (GA) is a natural gum that has been widely used as an emulsifying agent and shows promise for dispersing nanoparticles in aqueous solutions. The adsorption of GA onto HAp nanoparticles followed a Langmuir isotherm with an adsorption plateau occurring at 0.2 g GA/g HAp. The adsorption of GA onto MNP attained a maximum value of 0.6 g GA/g MNP, after which it decreased to approximately 0.2 g GA/g MNP. The maximum adsorption density of GA on both MNP and HAp is equivalent when normalized to the specific surface area (4 × 10− 3 g GA/m2). Adsorbed GA molecules were displaced from the surface of HAp and MNP in the presence of phosphate ions. © 2007 Elsevier B.V. All rights reserved. Keywords: Adsorption; Hydroxyapatite; Magnetic nanoparticles; Surface modification; Gum Arabic

1. Introduction Nature has an extraordinary way of constructing elaborate composite structures with exceptional properties from relatively simple compounds. Unique concepts such as hierarchical nested design and self assembly phenomena are utilized in fascinating ways to synthesize biological composite systems by the controlled nucleation of an appropriate inorganic phase in the presence of a functionalized organic matrix. The matrix exerts a high degree of control over the development of the inorganic phase by regulation of ion flux at the matrix interface, growth and morphology modification of the inorganic phase by adsorption interactions with soluble polymer macromolecules, and functional group mediation of molecular interactions at the polymer/matrix interface [1]. Biomineralization processes result in elaborate, multifunctional architectures that display an extraordinary degree of control over the phase distribution, size, interfacial structure, morphology, and crystallographic orienta⁎ Corresponding author. Tel.: +1 202 319 5822; fax: +1 202 319 5195. E-mail address: [email protected] (O.C. Wilson). 1 Current address — REQUIMTE/CQFB, Centro de Química Fina e Biotecnologia, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal. 0928-4931/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2007.04.009

tion of the inorganic phase. Hydroxyapatite (HAp) and magnetic nanoparticles (MNP) are two of the most versatile bioceramic based nanoparticles that form in nature via biomineralization processes and have been adapted for use in various research and developmental efforts. HAp is the calcium based mineral reinforcing phase in mammalian hard tissue. HAp is widely used for protein purification and in the development of materials to restore damaged bone. Due to its important role in biomineralized hard tissues, most bone healing and remodeling therapies have focused on the preparation of HAp–polymer composites with the proper mechanical properties and bioactivity [2]. Some examples of HAp–polymer composites include HAp–gelatin [3], HAp–chitosan [4], HAp– collagen [5,6], HAp/collagen–alginate [7], HAp–silica [8], and HAp–polypropylene [9]. Polymeric matrix composites, which are bioinert, biodegradable and potentially injectable, can be particularly useful in hard tissue engineering [2]. Magnetic nanoparticles (both maghemite (γ-Fe2O3) and magnetite (Fe3O4)), possess many industrial and biomedical applications in powder or composite form. MNP are used for in vivo hyperthermia treatments for cancer, drug delivery, diagnostic-magnetic resonance imaging (MRI) and for in vitro separations, magnetorelaxometry, transfection, cell counting, and magnetic ELISA [10–17]. Magnetic polymer particles

444

A.C.A. Roque, O.C. Wilson Jr. / Materials Science and Engineering C 28 (2008) 443–447

dispersed in synthetic (e.g. polyvinyl alcohol, polyacrylates, polyesters, silica) and natural matrices (e.g. starch, pectin, dextran and alginates) have been synthesized and utilised since the mid-1970s [18–23]. The promising applications of HAp and MNP in both particulate and nanocomposite form drive the search for and development of surface modification agents that confer unique properties for targeted biological interactions and enhance colloid stability [5,18,21]. The tendency for nanoparticles in suspension to agglomerate complicates the processing of stable nanoparticle suspensions and nanocomposites with a high degree of particle dispersion. There are many surface modification agents that are able to improve the colloidal stability of nanoparticles through electrostatic, steric, or electrosteric effects but the chemistry of some dispersants make them unsuitable for use in biomedical applications. Gum Arabic (GA) is a unique dispersant that has been the subject of renewed interest as a dispersant for carbon nanotubes [24,25]. GA is a natural gum that is obtained from the acacia Senegal tree. GA possesses the ability to emulsify liquid–liquid and stabilize solid–liquid dispersions [26]. It has been previously noted that the adsorption of GA in oxide dispersions (α-Al2O3, TiO2, ZrO2 [27] and Fe3O4 [28]) can be explained based on electrostatic interactions between negatively charged groups from GA and positive sites on the oxide surface. GA is a highly heterogeneous material with branched structures, appearing as mixed calcium, magnesium and potassium salt of a polysaccharide acid. GA is composed of six carbohydrate moieties (galactopyranose, arabinopyranose, arabinofuranose, rhamnopyranose, glucopyranosyl uronic acid and 4-O methyl glucopyranosyl uronic acid) and a small portion of hydroxyproline rich protein [27]. GA can be regarded as an arabinogalactan-protein (AGP) complex. One of the more interesting attributes of GA is the fact that it exhibits similarities with extracellular matrix proteins and this feature may be useful in enhancing the behaviour of bioceramics. In this research work, we aimed to study the adsorption of GA on the surface of MNP and HAp nanoparticles using the bicinchoninic acid (BCA) assay. The BCA assay is a new technique for quantifying gum Arabic in aqueous solutions. This study demonstrates how gum Arabic, a naturally occurring emulsifier, can be used as a surface modification agent for HAp and MNP. We also investigated the influence of sodium phosphate on GA interactions with MNP and HAp in order to assess the stability of the adsorbed GA under physiological conditions. This competitive adsorption study is important considering the potential in vivo applications of GA encapsulated particles.

2.2. Determination of Gum Arabic (GA) in aqueous solutions A GA stock solution (150 mg/ml or 13 wt %) was prepared by slowly dissolving 30 grams of GA in distilled water (200 ml). This solution was centrifuged for 20 min at 3000 rpm (Sorvall RT centrifuge, model 6000B) and the supernatant was retained. The pelleted residue (0.84 g) was attributed to higher molecular weight moieties of GA. The GA stock solution was therefore defined as 145.8 mg/ml or 12.72 wt.% and serial dilution of this stock solution was used to prepare the standard GA solutions. The protein BCA test was used to determine the amount of GA in solution for the standard GA solutions by the following procedure. Fifty (50) μl of each GA sample was added to a well of a 96 well microplate (Costar, cell culture plates) and subsequently 200 μl of the BCA working reagent (50:1 volume ratio mixture of Reagent A: Reagent B) was added. The plate was incubated for 30 min at 37 °C and then the absorbance was read at 550 nm using a BioRad microplate reader (Benchmark). The absorbance data from the GA standard solutions was used to generate calibration curves (see Fig. 1). 2.3. Synthesis of magnetic nanoparticles (MNP) The MNP were synthesized via the Massart method with a few modifications [14]. A 2 l flask containing 450 ml of an aqueous 0.7 M NH4OH solution was flushed with nitrogen (N2) for 15 min. The reaction vessel was constantly stirred at approximately 1800 rpm using an external paddle-type stirrer. A solution (500 ml) containing 162 g of FeCl3 and 99 g of FeCl2 (1:2 molar ratio of Fe2+ to Fe3+) in deionised water (239 g) was then slowly added to the alkaline solution. It was necessary to add more (150 ml) of reagent ammonium hydroxide solution in order to maintain an alkaline pH above 10. The MNP suspension was allowed to stir for 1 hour and stored in polypropylene bottles at room temperature. The MNP suspension was washed via gravity sedimentation, removal of the supernatant, and redispersion of the MNP with deionised water. This procedure was repeated five times to wash excess ammonia from the particle suspension.

2. Experimental section 2.1. Materials The GA used in this work was donated by TIC Gums (Belcamp, MD). The protein BCA test reagents were obtained from Pierce Biotechnology (Rockford, IL). Chemicals for the synthesis of the magnetic nanoparticles (NH4OH, FeCl3 and FeCl2) were obtained from Sigma-Aldrich (St. Louis, MO). Nanophase HAp was synthesized according to Borum and Wilson [29].

Fig. 1. Adsorption profiles of gum Arabic onto magnetic nanoparticles (◼) and hydroxyapatite nanoparticles ( ) obtained by the quantification of free gum Arabic in solution at equilibrium (BCA test). Number of replicates: 4.



A.C.A. Roque, O.C. Wilson Jr. / Materials Science and Engineering C 28 (2008) 443–447

2.4. Characterization of MNP and HAp Physical characteristics of the MNP and HAp were determined via transmission electron microscopy (TEM), dynamic light scattering (DLS), and BET N2 adsorption to determine the specific surface area. The HAp nanoparticles displayed, an elongated, lathlike morphology (20–150 nm in length) based on TEM analysis [8,29]. The MNPs exhibited primary particles approximately 6–10 nm in diameter with equiaxed morphology. Both the HAp and MNP displayed a great deal of particle agglomeration based on TEM analysis, dynamic light scattering analysis, and observations of the sedimentation behaviour of particle suspensions. The specific surface area of the MNP and HAp nanoparticles was determined by BET N2 adsorption using a Quantasorb 1200 instrument (Quantachrome, Boyntown, FL). The MNP and HAp had specific surface areas of 150 and 50 m2/g, respectively.

445

varied between 0.1–1000 mM. The samples were ultrasonicated for 30–60 s and left incubating for 2 h at room temperature under orbital agitation. The amount of GA in solution was quantified using the BCA test as described previously. 3. Results and discussion 3.1. Adsorption of GA on MNP and HAp

2.6. Displacement of adsorbed GA from MNP and HAp surfaces by phosphate solutions

Proteins and reducing sugars can be measured using the chromogen bicinchoninc acid (BCA) test to quantify the amount of copper reduced from Cu2+ to Cu+ [30]. Since GA is a glycoprotein mainly composed of reducing sugars, it should possess the ability to reduce copper. This presents a new technique for the quantitative analysis of GA. The calibration curve data showed a linear and reproducible correlation between the concentration of GA and the measured absorbance at 550 nm. The calibration curves for low (0–20 mg/ml) and high concentrations (30–150 mg/ml) of GA presented correlation factors of R2 = 0.9974 and R2 = 0.9991 (number of replicates: 6). The reproducible quantification of GA in aqueous solutions was utilised to determine the adsorption profile of GA on MNP and HAp (Fig. 1). The adsorption isotherm of GA onto HAp and MNP displayed different profile characteristics. The isotherm for HAp displayed a Langmuir shape with a maximum of 0.2 g GA adsorbed/g. The profile for the adsorption of GA on MNP displayed a maximum of 0.6 g GA adsorbed/g, after which the adsorbed amount decreased to a value of approximately 0.2 g GA/g with increasing concentrations of GA in solution at equilibrium. It is interesting to note that the maximum amount of GA adsorbed on both MNP (0.6 g/g) and HAp (0.2 g/g) differs by a factor of three. This corresponds to the difference in the specific surface areas for MNP (150 m2/g) and HAp (50 m2/g). Therefore, the maximum adsorption density of GA on MNP and HAp when expressed on the basis of specific surface area was equivalent and equal to 0.004 g GA/m2. The difference in adsorption behaviour for GA interactions with MNP and HAp surfaces can be attributed to several factors. MNP and HAp particles differ in physical characteristics such as particle size, particle size distribution, and specific surface area. In addition, the surface chemistry of the MNP and HAp is

One ml of a standard 40 mg/ml GA solution was added to 10− 5 mol of the HAp and MNP suspensions, sonicated for 30– 60 s at a maximum power of seven watts, and equilibrated overnight at room temperature under orbital agitation. The aged samples were centrifuged for 10 min (HAp) and 20 min (MNP) at 14,000 rpm. The BCA test was used to quantify the amount of free GA in a 50 l aliquot of the supernatant solution and the remaining supernatant was carefully removed with a micropipette and discarded. The loosely adsorbed gum Arabic was washed from the particle surface by adding 500 μl of distilled water to the particles and centrifuging for 5 min (HAp) and 10 min (MNP) at 14,000 rpm. The supernatant was discarded (50 μl taken to quantify in the BCA test) and 1 ml of a phosphate solution (pH 7.5) containing 150 mM NaCl as a background electrolyte. The phosphate solution concentration was

Fig. 2. Displacement of adsorbed gum Arabic from the surface of magnetic and hydroxyapatite particles as a function of sodium phosphate solution concentration.

2.5. Adsorption of gum Arabic on magnetic and hydroxyapatite nanoparticles The concentration of nanoparticles in the stock suspensions was determined by drying 0.5 ml of the suspension at 80–90 °C and determining the mass of the nanoparticle residue. Aqueous suspensions of HAp (29.6 mg/ml) and MNP (4.2 mg/ml) were prepared for the adsorption experiments. Known volumes of the GA standard solutions (typically 1 ml) were added to nanoparticle suspensions containing 10– 4 mol of HAp and MNP and sonicated for 30–60 s at a maximum power of seven watts (Sonic Dismembrator 60, Fisher Scientific). The nanoparticle/ GA suspensions were allowed to equilibrate overnight at room temperature under orbital agitation. The aged samples were centrifuged for 10 min (HAp) and 20 min (MNP) at 14,000 rpm (Centrifuge 5415C, Eppendorf). Aliquots of the supernatant solutions (50 μl) were used to quantify the amount of free gum Arabic in equilibrium by the BCA test. The control experiment corresponded to the incubation of the nanoparticles with distilled water.

446

A.C.A. Roque, O.C. Wilson Jr. / Materials Science and Engineering C 28 (2008) 443–447

different and unique. Although both possess surface hydroxyl groups, the HAp has additional surface functional groups such as PO43− and CO32−. Gum Arabic is a high molecular weight polyelectrolyte macromolecule with charged groups that have a more anionic character. GA assumes a very compact structure with minimal chain–chain interactions and can fold on itself by utilising its own intramolecular hydrogen bonding [27]. It has been proposed that the glycoprotein moieties of GA contain negatively charged carboxylate groups that are capable of strong electrostatic and hydrogen-bond interactions with positively charged surface sites on oxide surfaces [27]. A possible explanation for the decrease in GA adsorption density on MNP at higher GA concentrations involves loosely bound GA surface molecules. It is feasible for GA to form a multilayer at the surface of MNP by intermolecular hydrogen bonding between adsorbed and free GA molecules. At high concentrations of GA, the molecules may start self-associating in solution and therefore shift the equilibrium established between the free and adsorbed GA molecules and only those that are effectively hydrogen bonding with free –OH groups on the MNP surface will remain adsorbed. 3.2. Displacement of adsorbed GA by phosphate ions The adsorption of GA at the surface of MNP and HAp is mainly due to electrostatic and hydrogen-bond type interactions between the exposed surfaces, involving carboxylic groups (glycoprotein carboxylate groups) and OH groups at the surface of the particles. When the electrostatic potential of the surfaces is crucial for the interaction, calcium, phosphate and hydrogen ions should play a very important role on the uptake of GA on these particles. In addition, when envisioning the use of these composites for in vivo applications, it is important to test the stability of the composite under usual cellular conditions. For this reason, we performed preliminary studies on the competitive adsorption of GA on MNP and HAp in the presence of phosphate ion using phosphate buffered saline solutions at constant neutral pH (Fig. 2). The displacement of GA from MNP nanoparticles increased with increasing concentrations of phosphate ions, with 45% of the adsorbed GA being released at physiological conditions (10 mM phosphate). The maximum amount of GA displaced corresponded to 50% of the initial amount adsorbed at a concentration of 1 M sodium phosphate. As for the HAp particles, about 25% of adsorbed GA seems to leave the surface in the presence of buffers with 0 and 0.1 mM sodium phosphate concentrations. At 1 mM sodium phosphate concentration, the percentage of GA released drops to 4%. After this point there was an increase in the amount of GA released with increasing sodium phosphate concentration. The percentage of GA that desorbed at 10 mM (physiological conditions) was 20% and 50% for 1M sodium phosphate concentrations. 4. Conclusion In this study we have explored a new agent for the stabilisation and surface modification of two distinct substrates with mul-

tifaceted applications in biomedicine. A new method to quantify gum Arabic in solution using the bicinchoninic acid test has been demonstrated and utilised to assess the adsorption of this agent at the surface of HAp and MNP. As expected, the adsorption mechanisms of GA onto these surfaces followed different adsorption plots, also reflected in the maximum amount of GA adsorbed. The displacement of the adsorbed GA in the presence of phosphate ions, which naturally exist in biological fluids, was also tested showing 50% removal of GA for concentrations of phosphate two orders of magnitude higher than the normal physiological values. This preliminary work shows the potential applications of gum Arabic as a surface modification agent for HAp and MNP and encourages further studies to develop interesting applications for these composites in biomedicine and biotechnology. Acknowledgements A.C.A. Roque acknowledged a research grant from Fundação Calouste Gulbenkian (Portugal). The authors would like to thank Dr. Darryl Williams (University of Maryland), Dr. Patrick Mehl (CUA) and Dr. Michael Mullins (CUA) for their valuable contributions to this work. References [1] P. Calvert, S. Mann, J. Mater. Sci. 23 (1988) 3801. [2] J.F. Mano, R.A. Sousa, L.F. Boesel, N.M. Neves, R.L. Reis, Compos. Sci. Tech. 64 (2004) 789. [3] M.C. Chang, C.C. Ko, W.H. Douglas, Biomaterials 24 (2003) 2853. [4] I. Yamaguchi, K. Tokuchi, H. Fukuzaki, Y. Koyama, K. Takakuda, H. Monma, J. Tanaka, J. Biomed. Mater. Res. 55 (2001) 20. [5] M. Kikuchi, H.N. Matsumoto, T. Yamada, Y. Koyama, K. Takakuda, J. Tanaka, Biomaterials 25 (2004) 63. [6] M. Kikuchi, T. Ikoma, S. Itoh, H.N. Matsumoto, Y. Koyama, K. Takakuda, K. Shinomiya, J. Tanaka, Compos. Sci. Technol. 64 (2004) 819. [7] S. Sotome, T. Uemura, M. Kikuchi, J. Chen, S. Itoh, J. Tanaka, T. Tateishi, K. Shinomiya, Mater. Sci. Eng., C 24 (2004) 341. [8] L. Borum, O.C. Wilson Jr., Biomaterials 24 (2003) 3681. [9] M. Bonner, I.M. Ward, W. McGregor, K.E. Tanner, W. Bonfield, J. Mater. Sci. Lett. 20 (2001) 2049. [10] C.C. Berry, A.S.G. Curtis, J. Phys. D Appl. Phys. 36 (2003) R198. [11] M. Shinkai, M. Yanase, M. Suzuki, H. Honda, T. Wakabayashi, J. Yoshida, T. Kobayashi, J. Magn. Magn. Mater. 194 (1999) 176. [12] A. Jordan, R. Scholz, P. Wust, H. Schirra, T. Schiestel, H. Schmidt, R. Felix, J. Magn. Magn. Mater. 194 (1999) 185. [13] S.J. Lee, J.R. Jeong, S.C. Shin, J.C. Kim, Y.H. Chang, J.D. Kim, J. Magn. Magn. Mater. 272–276 (2004) 2432. [14] C. Xu, K. Xu, H. Gu, X. Zhong, Z. Guo, R. Zheng, X. Zhang, B. Xu, J. Am. Chem. Soc. 126 (2004) 3392. [15] P. Tartaj, M.P. Morales, S. Veitemillas-Verdaguer, T. Gonzalez-Carreno, C.J. Serna, J. Phys. D: Appl. Phys. 36 (2003) R182. [16] Q.A. Pankhurst, J. Connolly, S.K. Jones, J. Dobson, J. Phys. D: Appl. Phys. 36 (2003) R167. [17] V. Strom, K. Hultenby, C. Gruttner, J. Teller, B. Xu, J. Holgersson, Nanotechnology 15 (2004) 457. [18] C. Wilhelm, C. Billotey, J. Roger, J.N. Pons, J.C. Bacri, F. Gazeau, Biomaterials 24 (2003) 1001. [19] M. Mikhaylova, D.K. Kim, N. Bobrysheva, M. Osmolowsky, V. Semenov, T. Tsakalakos, M. Muhammed, Langmuir 20 (2004) 2472. [20] X. Teng, H. Yang, J. Mater. Chem. 14 (2004) 774. [21] C. Bergemann, D. Muller-Schulte, J. Oster, L. Brassard, A.S. Lubbe, J. Magn. Magn. Mater. 194 (1999) 45.

A.C.A. Roque, O.C. Wilson Jr. / Materials Science and Engineering C 28 (2008) 443–447 [22] L.M. Lacava, V.A.P. Garcia, S. Kuckelhaus, R.B. Azevedo, N. Sadeghiani, N. Buske, P.C. Morais, Z.G.M. Lacava, J. Magn. Magn. Mater. 272–276 (2004) 2434. [23] C. Gruttner, J. Teller, J. Magn. Magn. Mater. 194 (1999) 8. [24] R. Dagani, Chem. Eng. Newsl. 80 (28) (2002) 38. [25] R. Bandyopadhyaya, E. Nativ-Roth, O. Regev, R. Yerushalmi-Rozen, Nano Lett. 2 (2002) 25. [26] A.M. Islam, G.O. Phillips, A. Sljivo, M.J. Snowden, P.A. Williams, Food Hydrocoll. 11 (1997) 493.

447

[27] Y.K. Leong, U. Seah, S.Y. Chu, B.C. Ong, Colloids Surf. A Physicochem. Eng. Asp. 182 (2001) 263. [28] D.N. Williams, K.A. Gold, T.R. Pulliam-Holoman, S.H. Ehrman, O.C. Wilson Jr., J. Nanopart. Res. 8 (2006) 749. [29] L. Borum, O.C. Wilson Jr., Biomaterials 24 (2003) 3671. [30] G.E. Anthon, D.M. Barrett, Anal. Biochem. 305 (2002) 287.