International Journal of Pharmaceutics 477 (2014) 553–563
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Bionanocomposites containing magnetic graphite as potential systems for drug delivery Lígia N.M. Ribeiro a,b,c , Ana C.S. Alcântara a,1, Margarita Darder a , Pilar Aranda a , Paulo S.P. Herrmann c, Fernando M. Araújo-Moreira b , Mar García-Hernández a , Eduardo Ruiz-Hitzky a, * a
Instituto de Ciencia de Materiales de Madrid, CSIC, Cantoblanco, 28049 Madrid, Spain Universidade Federal de São Carlos (UFSCar), Departamento de Física, Rodovia Washington Luis Km 235, Monjolinho,13565-905 São Carlos, SP, Brazil c Embrapa Instrumentação Agropecuária, CP 741, 13560-970 São Carlos, SP, Brazil b
A R T I C L E I N F O
A B S T R A C T
Article history: Received 21 July 2014 Received in revised form 7 October 2014 Accepted 11 October 2014 Available online 16 October 2014
New magnetic bio-hybrid matrices for potential application in drug delivery are developed from the assembly of the biopolymer alginate and magnetic graphite nanoparticles. Ibuprofen (IBU) intercalated in a Mg–Al layered double hydroxide (LDH) was chosen as a model drug delivery system (DDS) to be incorporated as third component of the magnetic bionanocomposite DDS. For comparative purposes DDS based on the incorporation of pure IBU in the magnetic bio-hybrid matrices were also studied. All the resulting magnetic bionanocomposites were processed as beads and films and characterized by different techniques with the aim to elucidate the role of the magnetic graphite on the systems, as well as that of the inorganic brucite-like layers in the drug-loaded LDH. In this way, the influence of both inorganic components on the mechanical properties, the water uptake ability, and the kinetics of the drug release from these magnetic systems were determined. In addition, the possibility of modulating the levels of IBU release by stimulating the bionanocomposites with an external magnetic field was also evaluated in in vitro assays. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Bionanocomposites Magnetic graphite Layered double hydroxides Drug delivery Alginate Ibuprofen Controlled release
1. Introduction Nowadays, numerous studies are focused on the development of bio-hybrid systems, including those denoted as bionanocomposites that are based on biopolymers and nanoparticulated inorganic solids, with the aim of exploiting their properties for an extensive range of applications (Darder et al., 2007; Ruiz-Hitzky et al., 2008a,b, 2010a,b,c; Ruiz-Hitzky and Fernandes, 2013a,b). These type of materials are especially interesting in the biomedical field thanks to their safety and biocompatibility, being applied from wound dressing and tissue engineering to drug delivery (Fernandes et al., 2013; Lvov and Abdullayev, 2013; Park et al., 2013; Ruiz-Hitzky et al., 2008a, 2010b, 2013). The advance in the drug delivery systems (DDS) based on bionanocomposite materials provides materials with efficient chemical or physical barriers to
* Corresponding author. Tel.: +34 913349000; fax: +34 913720623. E-mail address:
[email protected] (E. Ruiz-Hitzky). 1 Present address: Universidade Federal do Rio Grande do Norte, UFRN, Departamento de Química, LABPEMOL, Lagoa Nova, 59072-970 Natal, RN, Brazil. http://dx.doi.org/10.1016/j.ijpharm.2014.10.033 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
control the speed of the drug release and the maintenance of the desired dose, combined with a simple, cheap, versatile and biocompatible synthesis process (Prabaharan and Jayakumar, 2011). The biocompatible character of biopolymers such as polysaccharides or proteins has been widely profited for application in the controlled release of drugs (Alvarez-Lorenzo et al., 2013; Coviello et al., 2007; Liu et al., 2005; Luo et al., 2011; Pongjanyakul and Puttipipatkhachorn, 2007; Young et al., 2005). Among the polysaccharide group, alginate is commonly used in the development of DDS. It is a linear polysaccharide comprised of a-L-guluronic acid and b-D-mannuronic acid, which is extracted from brown seaweeds. It can be easily processed as beads, films or foams for many different applications. Alginate presents an advantageous property, as it can form a gel by crosslinking reactions with divalent cations, such as Ca2+ and Zn2+, decreasing its solubility and often improving other properties such as mechanical resistance (Pongjanyakul and Puttipipatkhachorn, 2007). These types of reactions are very useful in the preparation of beads as well as in the stabilization of films or foams for the more diverse applications. Alginate-based bionanocomposites applied as DDS usually incorporate a hybrid
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material prepared by previous intercalation of the drug in a layered substrate such as clays (Fan et al., 2013; Iliescu et al., 2014) and double hydroxides (LDH), with the aim of increasing the stability of the system in order to procure an increased control over the release of the entrapped drug (Alcântara et al., 2010; Zhang et al., 2010). Layered double hydroxides (LDHs) consisting of brucite-like layers composed by hydroxides of divalent and trivalent metal cations, are commonly used as the inorganic counterpart of bio-hybrid systems for DDS purposes, due to their large anion exchange capacity and biocompatibility (Ruiz-Hitzky et al., 2010c). Besides a wide variety of drugs (Ambrogi et al., 2003; Costantino et al., 2009, 2012; Choy et al., 2004; Gordijo et al., 2005; Oh et al., 2012; Ribeiro et al., 2014; Tyner et al., 2004), several types of biomolecules have been intercalated into the brucite-like layers of LDHs, such as the anticoagulant heparin (Gu et al., 2008), phenylalanine and other amino acids (Aisawa et al., 2004) or even DNA (Choy et al., 1999). Exclusive properties can be achieved by incorporating magnetic nanoparticles into the biopolymer matrices (Schexnailder and Schmidt, 2009). Actually, a large number of magnetic materials have been used for abundant technological and biomedical applications, including magnetic separation, MRI contrast agent, hyperthermia, thermal ablation and tissue engineering (Kim et al., 2012; Meenach et al., 2010; Reddy et al., 2011; Tartaj et al., 2003). For the DDS area, this is one of the most interesting purposes, especially for cancer therapy, where the levels of drug release can be tuned through stimulation by an external magnetic field (Chomoucka et al., 2010). In this context, LDH-drug containing materials provided with magnetic properties typically containing iron-oxide particles are currently explored in view to DDS applications (Ay et al., 2011; Pan et al., 2011; Wang et al., 2010). The incorporation of magnetic properties to LDH-based DDS can be used to procure a targeted controlled delivery of the drug as recently pointed out by Huang et al. (2013). The major drawback of many of the abovementioned magnetic DDS is related to the use of toxic reagents (e.g., organic solvents, initiators or surfactants), frequently required in the synthesis process or in the preparation of the nanoparticles dispersion, as they are undesirable for biomedical applications. In this sense, novel magnetic graphite nanoparticles patented by AraújoMoreira and Mombrú groups (Araújo-Moreira et al., 2005) were produced by a cheap and simple process based on the oxidation– reduction reaction of pristine graphite with controlled amounts of oxygen released from the decomposition of CuO at high temperature, allowing to obtain macroscopic amounts of magnetic pure graphite nanoparticles (Faccio et al., 2008; Mombrú et al., 2005; Pardo et al., 2006, 2012; Souza et al., 2012). These nanoparticles synthesized according to predetermined parameters (Araújo-Moreira et al., 2005) can be easily dispersed in bidistilled water only by sonication, in the absence of the usually required surfactants, organic solvents or initiators. Thus, this carbonaceous material may be potentially used in a broad range of applications, especially for biomedical purposes such as tissue engineering, hyperthermia and DDS. In this work, we propose the development of magnetic biohybrid matrices based on the combination of alginate and magnetic graphite nanoparticles, in which the nonsteroidal antiinflammatory drug ibuprofen (IBU) alone or intercalated in a Mg– Al layered double hydroxide (LDH) was chosen as the third component of the new type of magnetic bionanocomposite DDS. It is expected that the system involving the biopolymer and the two inorganic solids will combine three main advantages: (i) the protection afforded by the biocompatible alginate and its ability to be prepared as beads and films, (ii) the presence of magnetic graphite nanoparticles that improve the physical and mechanical
properties of the biopolymer and afford hydrophobic and magnetic character, and (iii) the protective effect of the LDH layers entrapping the IBU molecules that slow down the release rate of the drug. For confirming these assumptions, in vitro tests were carried out in order to evaluate the behavior of these new magnetic bionanocomposite materials in the controlled delivery of the entrapped ibuprofen. 2. Experimental 2.1. Starting materials and reagents Alginate (ALG) and sodium ibuprofen (IBU) were purchased from Sigma–Aldrich. Aqueous solutions were prepared from chemicals of analytical reagent grade: AlCl36H2O (>99%, Fluka), MgCl26H2O (Panreac), NaOH (98%, Fluka), NaCl (>99%, Sigma– Aldrich), Na2CO3 (>99%, Merck), NaH2PO4H2O (>99%, Sigma–Aldrich) and CaCl2 2H2O (>99%, Sigma–Aldrich). Bidistilled water (resistivity of 18.2 MV cm) was obtained with a Maxima Ultrapure Water from Elga. Magnetic graphite (MG) was synthesized as described elsewhere (Araújo-Moreira et al., 2005). 2.2. Synthesis of the layered double hydroxide/ibuprofen intercalation compound The intercalation compound of IBU in a [Mg0.67Al0.33(OH)6] Cl0.33nH2O (Mg2Al-chloride) LDH was synthesized by co-precipitation method at constant pH, following the procedure described by Constantino and Pinnavaia (1995). A mixture of MgCl26H2O (18 mmol) and AlCl3.6H2O (9 mmol) was dissolved in 400 mL of decarbonated bidistilled water. This aqueous solution and 1 M NaOH solution were added dropwise to 100 mL of aqueous solution containing 0.5 g IBU using a DOSINO pH module 800 (Metrohm). This system permitted a controlled addition of solutions in order to maintain a constant pH around 9.0 during the synthesis. The resulting suspension was kept under nitrogen flow to remove CO2 and under magnetic stirring for 48 h at 60 C. The solid product was isolated by centrifugation, washed several times with bidistilled water, and dried overnight at 60 C. The resulting hybrid material was denoted as LDH–IBU. 2.3. Preparation of alginate–magnetic graphite bionanocomposite beads Beads of the alginate–magnetic graphite bionanocomposites were prepared according to the following procedure: an aqueous solution of 2% (w/v) alginate was magnetically stirred with 1% (w/ v) of pure IBU or the necessary amount of LDH–IBU intercalation compound containing 1% (w/v) IBU until homogenization. Then, a stable dispersion of 0.004% (w/v) magnetic graphite in bidistilled water was added to the alginate solution and homogenized by sonication. This mixture was introduced in a burette and then slowly poured as small droplets into a solution of 15% CaCl2 for 3 h. The resulting beads were filtered and washed with abundant doubly distilled water to remove non-entrapped IBU and residual Ca2+ ions, frozen in liquid nitrogen and lyophilized in a freezedryer (Cryodos, Telstar) for later use. The beads containing pure IBU were denoted as ALG/IBU/MG, and those containing LDH–IBU as ALG/LDH–IBU/MG. For comparison, other batches without magnetic graphite nanoparticles were prepared following the same procedure except the addition of the magnetic graphite suspension. The resulting beads were denoted as ALG/IBU when incorporating IBU alone and as ALG/LDH–IBU when the hybrid was added. The initial amount of IBU and MG per gram of alginate in the preparation of the alginate-based beads are summarized in Table 1.
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Table 1 Initial amount of ibuprofen (IBU) and magnetic graphite (MG) per gram of alginate used in the preparation of the alginate-based beads. Compositions of samples
Nomenclature of samples
IBU (g)
MG (g)
Alginate encapsulating IBU Alginate–magnetic graphite encapsulating IBU Alginate encapsulating LDH-intercalated IBU Alginate–magnetic graphite encapsulating LDH-intercalated IBU
ALG/IBU ALG/IBU/MG ALG/LDH–IBU ALG/LDH–IBU/MG
o.1 0.1 0.1 0.1
0 0.002 0 0.002
2.4. Preparation of alginate–magnetic graphite bionanocomposite films Films of the alginate-based bionanocomposites with different formulations were produced from the alginate and alginate/MG suspensions loaded with IBU or LDH–IBU, described in the previous section, by means of a casting/solvent evaporation technique. The different gels were poured in Petri dishes and allowed to dry for 72 h at room temperature. Then, the films were gently removed and cross-linked by 5% (w/v) CaCl2 for 15 min, washed and finally dried at room temperature. 2.5. Characterization Powder X-ray diffraction (XRD) data were collected in a Bruker D8-ADVANCE diffractometer using a CuKa source, with a scan step of 2 min1 between 2u values of 2 and 70 . Fourier transfer infrared (FTIR) spectra were recorded with a FTIR spectrophotometer Bruker IFS 66 v/S. Each sample was placed in the sample holder as KBr pellets and scanned from 4000 to 250 cm1 with 2 cm1 resolution. The amount of organic matter in the LDH intercalation compound was determined by CHN elemental chemical microanalysis in a PerkinElmer 2400 series II CHNS/O elemental analyzer. The thermal behavior of the diverse prepared materials was analyzed from the simultaneously recorded thermogravimetric (TG) and differential thermal analysis (DTA) curves in a SEIKO SSC/5200 equipment, in experiments carried out under air atmosphere (flux of 100 mL min1) from room temperature to 1000 C at 10 C min1 heating rate. Surface morphology was observed in a ZEISS DSM-960 microscope working at 15 kV and FE-SEM equipment FEI-NOVA NanoSEM 230. The magnetic behavior of the samples was characterized using a SQUID magnetometer from Quantum Design, equipped with a 5 T coil. Magnetization vs. temperature were conducted following a zero field cool-field cool (ZFC-FC) protocol (H cool = H measure = 1000 Oe). The isothermal field dependence of the magnetization was measured at 5.0 K and 300 K. The diamagnetic contribution was calculated from the high field slope of the 300 K hysteresis cycle and subtracted from the isotherms and from the M vs. Temperature curves.
the original length). The mechanical properties were analyzed as a function of magnetic graphite present in the batches. All the experiments were carried out five times. 2.7. Estimation of IBU loading and encapsulation efficiency of the beads Weighed bionanocomposite beads (0.1 g) were immersed in 50 mL of 2 M phosphate buffer pH 7.4 for 12 h. Then, the solution was filtered and the IBU content was calculated from the absorbance measured at l = 262 nm with a UV–vis spectrophotometer (Shimadzu UV-2401), using quartz cuvettes with a 1 cm path length (Babu et al., 2006). The estimation of the drug percentage loading and the encapsulation efficiency were obtained using Eqs. (1) and (2) (Ambrogi et al., 2008). All the experiments were carried out in triplicate. %drug loading ¼
amount of drugin beads 100 amount of beads
%encapulation efficiency ¼
drug loading 100 theoretical loading
(1)
(2)
In Eq. (2), the theoretical loading refers to the initial amount of ibuprofen used in the preparation of the beads indicated in Table 1. 2.8. Water uptake studies Aliquots of each system of bionanocomposite beads (0.05 g) were placed in a Petri dish, immersed in 2 M phosphate buffer pH 7.4, and shaken eventually at room temperature. After a predetermined time interval, the beads were withdrawn and weighed on an analytical balance after removing the excess of water. The water uptake was calculated from Eq. (3) (RemuñánLopez and Bodmeier, 1997). This experiment was performed similarly for the films, with a mass of 0.05 g in all the batches. All the experiments were carried out in triplicate. water uptakeðmg=mgÞ ¼
W2 W1 W1
(3)
where W2 and W1 are the wet and initial mass of beads or films, respectively.
2.6. Mechanical properties 2.9. In vitro IBU cumulative release studies In order to verify the mechanical properties of dry and wet films, a stress–strain test was executed with a Model 1122 Instron Universal Testing Machine (Instron Corp., Canton, MA, USA). The films were cut to have rectangular dimensions (100 mm 5 mm) in accordance to the ASTM D882-97 test. The dry films were conditioned at 30% RH and 25 C for 48 h before measurements and the moisturized films were then immersed in bidistilled water for 2 h before the study. The maximum tensile strength (TS), maximum percentage elongation at break (EB%), and elastic modulus (or Young’s modulus) were determined. Films were stretched using a speed of 50 mm min1. Tensile properties were calculated from the plot of stress (tensile force/initial cross-sectional area) vs. strain (extension as a fraction of
In a standard study, bionanocomposite beads (0.1 g) were added to 50 mL of release medium (2 M phosphate buffer solution pH 7.4) and bionanocomposite films (1 cm 1 cm) with a mass of 0.1 g were added to 15 mL of the same release medium, both in thermostatic baths at 37 C. The release study was carried out for 8 h. After 30 min time intervals, 3 mL of each medium were withdrawn and the amount of IBU released from the drug-loaded materials was analyzed by UV spectrophotometry (l = 262 nm), and finally the measured sample was added back to the solution. In order to evaluate the drug release profile of the magnetic bionanocomposite under magnetic stimulation, a magnetic field
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(1 T) was applied during the release study. The magnet was positioned just beside the flask and was maintained unchangeable under the whole in vitro release process. All the experiments were carried out in triplicate. 3. Results and discussion 3.1. Synthesis and characterization of the bionanocomposite beads The bionanocomposites were prepared by dispersing the pure drug or the LDH–IBU hybrid in alginate, together with presynthesized magnetic graphite nanoparticles using 15% CaCl2 to produce the cross-linking of the biopolymer, affording the formation of magnetic bio-hybrid beads (Fig. 1). With the use of LDH–IBU it is intended to have a better control in the release rate of the drug as proposed by diverse authors (Alcântara et al., 2010; Costantino et al., 2009; Khan et al., 2001; Tyner et al., 2004). In the present case, the LDH–IBU hybrid was prepared by the coprecipitation method resulting in an intercalation compound containing 21.17% (w/w) of IBU, representing around 130 mEq per 100 g LDH. Given that the anion-exchange capacity of the LDH is about 330 mEq/100g, it can be deduced that the exchange reaction was not complete, and a reduced amount of chloride ions may remain in the interlayer space contributing to compensate the positive charge of the layers as has been also observed elsewhere (Alcântara et al., 2010; Gordijo et al., 2005). XRD and FTIR characterization confirm the presence of ibuprofen molecules in anionic form located in the interlayer region (basal spacing increase of 1.89 nm) therefore able to compensate the positive charge of LDH layers (see Supplementary information). Both the ALG/LDH–IBU bionanocomposite and the IBU-loaded alginate systems have been also implemented with magnetic graphite nanoparticles (MG) that show a ferromagnetic behavior, with a strong saturation magnetic moment close to 0.25 emu/g at 300 K (Araújo-Moreira et al., 2005; Mombrú et al., 2005). An important advantage of these graphite nanoparticles is their ability to form stable suspensions in bidistilled water by sonication,
Fig. 1. Scheme of the magnetic bionanocomposite system based on ALG/LDH–IBU/ MG.
without the necessity to add surfactants, organic solvents or acids as reported for other magnetic systems. Thus, MG can be easily dispersed within the biopolymer matrices providing the resulting bionanocomposites with magnetic properties together with hydrophobicity, which can have also a strong influence on the water sorption properties of these materials and, consequently, on the rate of drug release (see Section 3.3). Fig. 2a and b shows the ZFC-FC measurements of temperature dependence of the magnetization for both samples ALG/IBU/MG and ALG/LDH–IBU/MG, respectively. The main features, regarding the magnetism reported by Mombrú et al. for the pristine graphite sample (Mombrú et al., 2005), remain as the main ingredient of a multilevel development of the ferromagnetic behavior it is also observed in our samples. However some differences become apparent: first, the magnetic signal is strongly reduced with respect to that of the pristine magnetic graphite sample (Mombrú et al., 2005) since magnetic graphite is very diluted in our samples and the data have been normalized to the total mass; second, two main transitions are spotted for sample ALG/IBU/MG, now located at Tc1 = 237 K and Tc2 around 272 K. For sample ALG/LDH–IBU/MG, a clear transition can be reported at Tc2 = 273 K and a small anomaly is also detected at Tc1 = 45 K. It is worth mentioning the Tc2 is sensitive to the cooling protocol and a decrease of the critical temperature is observed in both samples when cooling in a magnetic field (Delta T approx. 4 K). Large irreversibility is also observed in both samples pointing out to substantial magnetic disorder, as it was reported for the pristine sample. Note that at room temperature both samples remain ferromagnetic (ferromagnetic hysteresis cycles not shown) up to the highest temperatures explored (350 K).
Fig. 2. Temperature dependence of the magnetization for (a) ALG/IBU/MG and (b) ALG/LDH–IBU/MG samples.
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Table 2 Amount of IBU loaded and encapsulation efficiency in all the batches prepared as bionanocomposite beads (data are mean S.D., n = 3)
Fig. 3. Hysteresis cycles measured at 5 K for (a) ALG/IBU/MG and (b) ALG/LDH–IBU/ MG.
Fig. 3a and b shows the hysteresis cycles measured at 5 K. As compared to the reported values for pristine graphite (Mombrú et al., 2005), the coercive fields are much smaller for both samples (Hc = 150 Oe for ALG/IBU/MG and Hc = 98 Oe for ALG/LDH–IBU/ MG). Both samples exhibit a clear ferromagnetic behavior with a finite remanence and a finite coercive field. The high field values of the magnetization are larger for ALG/LDH–IBU/MG than for ALG/ IBU/MG. None of the samples saturates as a small paramagnetic contribution is present at low temperatures. Irreversibility is larger for ALG/LDH–IBU/MG. In pristine graphite (Mombrú et al., 2005), the observed ferromagnetic behavior was ascribed to the existence of a variety of defects resulting from the vapor phase redox reaction of graphite in a N2 atmosphere. In this context, differences observed in the temperature and field dependence of the magnetizations in our samples, as compared to the pristine graphite, are to be explained in terms of changes in the number and distribution of defects as a consequence of the ulterior processing of the graphite particles into the matrix and also to the appearance of new interactions in the composite. The encapsulation efficiency of the alginate-based DDS and the percentage of drug loaded in different batches of beads with and without the incorporation of MG nanoparticles are listed in Table 2. It is observed that the amount of IBU loaded in the different beads is very similar when the drug is intercalated either directly or within the brucite-like layers, being higher when magnetic nanoparticles are present in both types of matrices. These results suggest that the presence of the MG contributes to increase the encapsulation efficiency, probably due to its hydrophobic character, which would prevent the leaching of the drug during the beads processing, leading to values about twice those of systems without magnetic particles.
Formulation
IBU loading (%)
Encapsulation efficiency (%)
ALG/IBU ALG/LDH–IBU ALG/IBU/MG ALG/LDH–IBU/MG
2.73 0.55 2.55 0.25 4.49 0.06 4.70 0.17
30.3 6.1 37.6 3.7 49.9 0.6 69.1 2.5
The FTIR spectra of the alginate–magnetic graphite bionanocomposites incorporating LDH–IBU and IBU are shown in Fig. 4. The spectrum of pristine alginate (Fig. 4a) reveals bands at 3363, 1609, 1414 and 1034 cm1, corresponding to nOH(H2O and OH), nasCO(—COO), nsCO(—COO) and nasCO(C—O—C) vibration modes, respectively (Alcântara et al., 2010). The spectrum of pure magnetic graphite (Fig. 4b) shows bands at 1038 and 1637 cm1, attributed to the dC—H and nC—C vibration modes, respectively. The other band at 3443 cm1 can be attributed to nOH vibration modes. The spectra of the alginate/MG (Fig. 4c) and the alginate/LDH–IBU/ MG (Fig. 4d) beads show a shift of the vibration bands observed in the alginate spectrum at 1414 and 1609 cm1 to higher wavenumber values in both cases, which is attributed to the existence of ionic interactions between Ca2+ ions and the carboxylate groups of alginate as a consequence of the cross-linking process introduced in the formation of the beads (Pongjanyakul and Puttipipatkhachorn, 2007). Besides the cross-linking effect, the existence of interactions between the carboxylate groups and the magnetic graphite nanoparticles should not be discarded, as observed in FTIR studies on the interaction of carbon nanotubes and sodium alginate previously reported (Zhao et al., 2009). On the other hand, the spectrum of alginate/LDH–IBU/MG beads (Fig. 4d) shows a low intensity band at 589 cm1 related to the O—M—O bending vibration mode of the brucite-like structural layers, which confirms the preservation of the LDH structure once incorporated in the bionanocomposite beads. The broad band centered at 3422 cm1 can be ascribed to the O—H vibrations of hydroxyl groups in alginate as well as in adsorbed water molecules. The TG curve of pure alginate (Fig. 5a) shows a mass loss associated with the elimination of adsorbed water molecules (9%) up to 100 C, followed by partial biopolymer decomposition and combustion centered at 311 C and 594 C, respectively (Soares et al., 2004). The curve of pristine magnetic graphite (Fig. 5b)
Fig. 4. FTIR spectra (4000–400 cm1 region) of pristine alginate (a), pristine magnetic graphite (b), ALG/MG (c) and ALG/LDH–IBU/MG (d) bionanocomposite beads.
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Fig. 5. TG and DTA curves obtained for: alginate (a), magnetic graphite (b), ALG/MG beads (c) and ALG/LDH–IBU/MG bionanocomposite beads (d), in air (flux of 100 mL min1).
exhibits three exothermic peaks around 352 C, 590 C and 628 C, which lead to a mass loss (29.5%) associated with partial decomposition of graphite. The TG curve of beads containing only ALG/MG (Fig. 5c) shows a mass loss associated with the elimination of adsorbed water molecules (16.7%) up to 64 C. Additionally, three exothermic events can be observed at 417 C, 437.5 C and 739 C, attributed to the partial decomposition and combustion of alginate, followed by the decomposition of the magnetic graphite, respectively. Furthermore, it can be observed in this curve that the temperature of alginate decomposition is higher than in the pristine alginate, possibly due to the interactions between both components (Travlou et al., 2013). A similar thermal profile was observed for ALG/LDH–IBU/MG beads (Fig. 5d), in which two mass losses of about 17.6% and 14% occur up to 200 C, being related to the loss of physically adsorbed water molecules and the combustion of the IBU molecules confined in the inorganic matrix. The DTA curve (Fig. 5d) shows two exothermic peaks, one at 395 C associated with the combustion of IBU molecules, and other around 450 C, which can be attributed to the alginate decomposition and combustion. Other thermal events at higher temperatures can be associated with the decomposition of the aromatic structural groups of IBU and MG. It is remarkable that in all the systems the MG presents an excellent thermal stability that may improve significantly the stability of the whole bio-hybrid system. It is also remarkable the protective effect of the inorganic layers in the LDH-IBU system. While the TG-DTA analysis of IBU shows two exothermic peaks at 414 and 465 C due to decomposition of the organic molecules (Fig. S3), the decomposition of IBU in the hybrid is shifted toward higher temperatures (567 C).
The surface of ALG/LDH-IBU without MG (Fig. 6a) and ALG/ LDH-IBU/MG beads (Fig. 6b) were observed by FE-SEM in order to ascertain a possible effect of the magnetic graphite nanoparticles in the textural characteristics of the final bionanocomposite beads. In a general way, all the beads revealed a similar morphology, showing homogeneity, roughness and certain porosity. In fact, the incorporation of MG seems not to affect the homogeneity observed in the ALG/LDH-IBU sample, showing a very similar aspect with an increased porosity. This suggests a satisfactory distribution of the MG particles as well as of the hybrid component within the biopolymeric matrix. 3.2. Physical properties of the bionanocomposite beads and films The mechanical properties of the developed bionanocomposite systems are clearly influenced by the presence of MG nanoparticles, as revealed by the stress–strain tests carried out on ALG/ IBU bionanocomposites with and without MG nanoparticles that were processed as films for these experiments. The study was carried out in dry and wet films, as the properties of the materials in the wet state can be different from those in the dry state (Remuñán-Lopez and Bodmeier, 1997). The values of maximum tensile strength (TS) and maximum percentage of elongation at break (EB%) for these materials are collected in Table 3. The TS values increase significantly with the presence of MG, being the dry ALG/IBU/MG films around 8 times more resistant than ALG/IBU films. Even in the case of wet films, which show a lower improvement than dry films, the TS values of wet ALG/IBU/MG are around 4 times higher than those of ALG/IBU films, confirming
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Fig. 6. FE-SEM images at different magnifications of the surface of ALG/LDH–IBU (a) and ALG/LDH–IBU/MG (b) bionanocomposite beads.
the contribution of magnetic graphite nanoparticles to improve the mechanical properties of the prepared films. The EB values of MGloaded films were also higher in both the dry and the wet states when compared with ALG/IBU films. The higher improvement of EB in the wet MG-loaded films can be attributed to the plasticizing effect of water, which increases the flexibility of the material, as reported by Remuñán-López in alginate cross-linked films (Remuñán-Lopez and Bodmeier, 1997). Such good properties observed here can be related to a possible interaction of carboxylate groups from the alginate with the magnetic graphite, as above mentioned in the FTIR studies, improving both mechanical properties and water absorption of the synthetized resistance towards bionanocomposites. The amount of water absorbed by the alginate–magnetic graphite bionanocomposites processed as beads and films increases with time of contact with phosphate buffer (pH 7.4), in all cases (Fig. 7). This aqueous medium was specifically chosen for the water uptake study because it is the simulated physiological medium in which the release study will be carried out. Fig. 7a and b shows the progress in the water content of all types of beads and films, respectively, as a function of time. The batches reached a constant content after around 6 h, except the films based on ALG/ IBU and ALG/LDH–IBU that were disintegrated before completion of the study. Table 3 Mechanical properties of alginate–magnetic graphite bionanocomposite films. Each value is the mean S.D. n = 5. P < 0.05. Formulation
ALG/IBU ALG/IBU/MG
Films in dry state
Films in wet state
TS (MPa)
EB (%)
TS (MPa)
EB (%)
7.99 1.46 63.26 9.25
4.77 1.43 6.63 1.76
2.03 0.33 8.61 2.30
4.77 0.65 18.75 5.69
Fig. 7. Water uptake of alginate-based bionanocomposite (a) beads and (b) films in phosphate buffer pH 7.4. Each value is the mean S.D. n = 3.
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In all cases the presence of phosphate ions in the simulated physiological medium can produce a release of the Ca2+ ions crosslinking the alginate chains, driving to the solubilization of the bionanocomposite films. However, the presence of MG and LDH in some samples seem to reduce the swelling ability and to increase the resistance towards disintegration, most likely due to their interaction with the polysaccharide chains and to the hydrophobicity shown by the MG nanoparticles. However, all the tested beads and the ALG/LDH–IBU/MG film swelled but did not dissolve during the water uptake study. This behavior might be related to a different degree of cross-linking in the samples when processed as beads or as films, as a consequence of a different concentration of CaCl2 and time of the cross-linking process in each case (RemuñánLopez and Bodmeier, 1997). In the case of the ALG/LDH–IBU/MG film, the presence of the hydrophobic MG nanoparticles may also contribute to slow down the water uptake process, besides the LDH acting as a physical barrier against water absorption. The incorporation of the IBU as a LDH–IBU hybrid seems to contribute to reduce the water uptake. Thus, the ALG/IBU bionanocomposite beads (Fig. 7a) reach a value of around 25.5 g of water per gram of bead, while the ALG/LDH–IBU bionanocomposite beads reach an adsorbed amount around 15.8 g, probably related to the higher stability of beads prepared with bionanocomposites containing the hybrid due to additional interactions with the biopolymer, as also observed in other LDH-based bionanocomposites (Alcântara et al., 2010; Aranda et al., 2012). On the other hand, a similar behavior was observed for the corresponding batches of ALG/IBU and ALG/LDH–IBU bionanocomposite films (Fig. 7b), absorbing around 12 g and 11 g of water/g of film, respectively, until their disintegration after about 3 h. The presence of magnetic graphite nanoparticles changes significantly the behavior of the bionanocomposites. Thus, the bionanocomposite beads containing MG exhibit in all cases lower water absorption values than the ones without MG. The ALG/IBU/ MG beads reached an uptake around 9 g/g of bead, about 16 g less than that ALG/IBU beads. This observation confirms the role of inorganic nanoparticles in the bionanocomposite in the increase of their stability towards water uptake. Thus, the ALG/LDH–IBU/MG beads presented the lowest water uptake, around 7 g of water per gram of beads, due to the presence of the hybrids and the magnetic graphite nanoparticles in the bionanocomposite. Similarly, ALG/ IBU/MG films absorbed only around 9 g of water in contrast to the 12 g per gram of film absorbed by the analogous ALG/IBU system without MG. The presence of MG nanoparticles also increased the
Fig. 8. Aspect of the (a) ALG/LDH–IBU and (b) ALG/LDH–IBU/MG bionanocomposite beads before and after immersion in phosphate buffer for 6 h.
stability of the bionanocomposites processed as films, as ALG/IBU films disintegrated after 3 h of study, while disintegration of ALG/ IBU/MG films occurred after 5 h. The water uptake of the ALG/LDH– IBU/MG bionanocomposite film was quite similar to that of the system incorporating IBU directly, reaching a water uptake of 10 g/ g of film. These results confirm the role of magnetic graphite in the bionanocomposites which not only improves the mechanical properties, but also confers more resistance to water absorption due to its hydrophobic character, acting as a physical barrier at the interface of the bionanocomposite and the medium, even in the batches without the LDH–IBU intercalation compounds. Fig. 8 shows the aspect of the beads before and after swelling in aqueous medium, with a considerable difference in their size due to the presence of the magnetic graphite nanoparticles. 3.3. In vitro release of ibuprofen from alginate–magnetic graphite bionanocomposites The medium of release of IBU, chosen as model drug in this study, was phosphate buffer pH 7.4 used as simulated physiological medium. Fig. 9 shows the release profile of all the studied batches and confirms that the presence of magnetic graphite nanoparticles in the beads slows down the rate of the drug delivery, providing a constant release during the whole experiment. The ALG/IBU and ALG/LDH–IBU beads rapidly reached around 99% of release after 3 and 4 h of study, respectively (Fig. 9a). These results show that DDS based on alginate are very sensitive to this medium, which facilitates the disintegration of the alginate matrix, speeding up the rate of IBU release after 150 min. However, those systems containing MG nanoparticles show a more controlled release profile during the experiment, mainly in the case of beads incorporating the drug within the LDH–IBU intercalation compound. For the ALG/IBU/MG and ALG/LDH–IBU/MG systems, the maximum released amount reached values around 89% and 68%, respectively, after 7 h of study. These results suggest that the hydrophobic magnetic graphite can act as a protective barrier, in accordance with the water uptake results. These factors control the
Fig. 9. Profile of IBU release from different bionanocomposite (a) beads and (b) films in phosphate buffer pH 7.4 at 37 C.
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passage of water molecules into the beads, providing a delay in the kinetics of release of the IBU into the medium. Moreover, ALG/ LDH–IBU/MG shows a slower release than ALG/IBU/MG, since the LDH-encapsulated IBU requires additional time to be released from the protective inorganic layered solid (Ribeiro et al., 2009). Fig. 9b shows the release behavior of IBU in the case of bionanocomposites processed as films. The ALG/IBU films released around 99% of the entrapped drug and the ALG/LDH–IBU films released around 95%, at 210 and 300 min of study, respectively. The ALG/LDH–IBU films reached a release slightly slower than ALG/IBU films because of the presence of the drug intercalated in the LDH host solid as mentioned above. The influence of MG nanoparticles in the bionanocomposite behavior was observed once more for the films. The ALG/IBU/MG sample shows a slightly slower rate of release than the systems without MG, and it took longer (about 6 h) to reach the total release of the drug. The ALG/LDH–IBU/MG film was again the bionanocomposite film that presented the more controlled release of the drug, reaching a release around 81% after 8 h of study. Generally, the bionanocomposites processed as films reached a higher release than those prepared as beads, because of several factors that can be related to a possible reduced degree of cross-linking in contact with the medium, facilitating the passage of water and salts into the matrices. Experimental kinetic data were fitted to various models such as those employed for release studies from alginate beads (Hwang et al., 1995) and LDH-based systems (Yang et al., 2007). Due to the complexity of the system here studied any of these models seems adequate to fit our results. This behavior may be related to the existence of simultaneous processes involving swelling, diffusion and beads disintegration. In the ALG/LDH–IBU/MG system the swelling behavior is modified by the presence of the hydrophobic MG and the whole process could be better adjusted to a zero-order model (Table 4). This model can be represented by the Eq. (4): Q t ¼ Q 0 þ k0 t
(4)
where Qt is the amount of drug dissolved in time (t), Q0 is the initial amount of drug in solution and k0 is the zero-order release constant. 3.4. Magnetically controlled release of ibuprofen The ALG/LDH–IBU/MG systems presented the most progressive and controlled IBU release of all the bionanocomposites, independently of their morphology, and were chosen to study their in vitro drug release under a magnetic stimulation by an external magnetic field. Thus, the IBU release profiles from ALG/LDH–IBU/MG bionanocomposite beads under the application of an external magnetic field and without it are shown in Fig. 10a. It was observed a clear change in the drug release behavior when the beads were stimulated. The maximum released amount reached values of around 88% with application of the external magnetic field, i.e., almost 20% higher than the IBU released from the beads without applying the magnetic field. A similar behavior is observed for the Table 4 Rate constant (k) and r2 coefficients (in parenthesis)c obtained based on zero-order equation from prepared formulation. Forms
Formulation
Zero-order (ka S.D.b ) (mg h1)
Beads
ALG/LDH–IBU/MG ALG/LDH–IBU/MG with magnet
19.49 0.718 (0.922) 25.26 1.146 (0.942)
Films
ALG/LDH–IBU/MG ALG/LDH–IBU/MG with magnet
19.51 0.974 (0.946) 23.66 0.769 (0.963)
a b c
Rate constant. Standard deviation is the mean. Coefficient of determination (r2).
561
Fig. 10. IBU release profile from ALG/LDH–IBU/MG bionanocomposite beads (a) and films (b) exposed or not to an external magnetic field (1 T), in phosphate buffer pH 7.4 at 37 C.
ALG/LDH–IBU/MG films (Fig. 10b), the magnetic stimulation was responsible for the improved levels of IBU release, reaching values around 95% after 8 h of study in contrast to the 81% obtained when the system was not magnetically stimulated. These events could be explained by a possible alignment of the magnetic moments of the magnetic graphite nanoparticles with that of the external magnetic field, which would lead to several alterations resulting in the expansion of the bionanocomposite network and allowing the release of higher amounts of the entrapped drug to the medium, as reported in the case of hybrid magnetic hydrogels (Giani et al., 2012). Finally, we can appreciate that the obtained rate constant (Table 4) is higher for the magnetically stimulated bionanocomposite. Efforts are in progress to completely elucidate the effects of the external magnetic field in order to understand the release mechanism, however the current results confirm that the application of the external magnetic field may modulate the levels of released IBU as it does the control of other parameters like contact time, temperature and pH, extensively reported in the literature. 4. Conclusions New bio-hybrid magnetic matrices based on alginate and magnetic graphite nanoparticles were developed incorporating ibuprofen as a model drug, either as the pure drug or as the LDH– IBU intercalation compound, and processed as beads or films for application as drug release systems. The presence of magnetic graphite nanoparticles improved the physical and mechanical properties of the resulting bionanocomposites, decreasing the speed of drug delivery due to the protective effect as a physical barrier against water absorption into the beads. The control on the release rate was specially improved when the drug was incorporated as the LDH–IBU intercalation compound, being this fact attributed to the additional physical barrier afforded by the inorganic layered host solid. These bionanocomposite systems could be also stimulated by an external magnetic field, enhancing
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the levels of the released ibuprofen, which would be advantageous in order to modulate the dose of released drug when required. This new magnetic DDS could be used for immobilization of other drugs and enzymes, but also as wound dressings or as scaffolds in tissue engineering, as they could be also processed as foams, for uses in controlled drug delivery. Acknowledgements This work was supported by the CICYT, Spain (project MAT2012-31759) and the EU COST Action MP1202. L.N.M.R. acknowledges the CAPES (Brazil) for her fellowship. FMAM acknowledges Brazilian agencies FAPESP, CAPES and CNPq for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. ijpharm.2014.10.033. References Aisawa, S., Kudo, H., Hoshi, T., Takahashi, S., Hirahara, H., Umetsu, Y., Narita, E., 2004. Intercalation behavior of amino acids into Zn–Al-layered double hydroxide by calcination–rehydration reaction. J. Solid State Chem. 177, 3987–3994. Alcântara, A.C.S., Aranda, P., Darder, M., Ruiz-Hitzky, E., 2010. Bionanocomposites based on alginate–zein/layered double hydroxide materials as drug delivery systems. J. Mater. Chem. 20, 9495–9504. Alvarez-Lorenzo, C., Blanco-Fernandez, B., Puga, A.M., Concheiro, A., 2013. Crosslinked ionic polysaccharides for stimuli-sensitive drug delivery. Adv. Drug Deliv. Rev. 65, 1148–1171. Ambrogi, V., Fardella, G., Grandolini, G., Nocchetti, M., Perioli, L., 2003. Effect of hydrotalcite-like compounds on the aqueous solubility of some poorly watersoluble drugs. J. Pharm. Sci. 92, 1407–1418. Ambrogi, V., Perioli, L., Ricci, M., Pulcini, L., Nocchetti, M., Giovagnoli, S., Rossi, C., 2008. Eudragit (R) and hydrotalcite-like anionic clay composite system for diclofenac colonic delivery. Microporous Mesoporous Mater. 115, 405–415. Aranda, P., Alcantara, A.C.S., Ribeiro, L.N.M., Darder, M., Ruiz-Hitzky, E., 2012. Bionanocomposites based on layered double hydroxides as drug delivery systems. In: Choi, H., Choy, J.H., Lee, U., Varadan, VK (Eds.), Proccedings SPIE, vol. 8548. Nanosystems in Engineering and Medicine #85486D. Araújo-Moreira, F.M., Pardo Minetti, H., Mombrú Rodríguez, A.W., 2005. A process of preparing magnetic graphitic materials, and materials there of. US Patent 8303836 B2. lu-Karan, B., 2011. Magnetic nanocomposites with Ay, A.N., Konuk, D., Zümreog drug-intercalated layered double hydroxide shell supported on commercial magnetite and laboratory-made magnesium ferrite core materials. Mater. Sci. Eng. C 31, 851–857. Babu, V.R., Rao, K., Sairam, M., Naidu, B.V.K., Hosamani, K.M., Aminabhavi, T.M., 2006. pH sensitive interpenetrating network microgels of sodium alginate-acrylic acid for the controlled release of ibuprofen. J. Appl. Polym. Sci. 99, 2671–2678. Constantino, V.R.L., Pinnavaia, T.J., 1995. Basic properties of Mg2+1xAl3+x layered double hydroxides intercalated by carbonate: hydroxide chloride and sulfate anions. Inorg. Chem. 34, 883–892. Costantino, U., Nocchetti, M., Sisani, M., Vivani, R., 2009. Recent progress in the synthesis and application of organically modified hydrotalcites. Z. Kristallogr. 224, 273–281. Costantino, U., Nocchetti, M., Tammaro, L., Vittoria, V., 2012. Modified hydrotalcitelike compounds as active fillers of biodegradable polymers for drug release and food packaging applications. Recent Pat. Nanotechnol. 6, 218–230. Coviello, T., Matricardi, P., Marianecci, C., Alhaique, F., 2007. Polysaccharide hydrogels for modified release formulations. J. Control. Release 119, 5–24. Chomoucka, J., Drbohlavova, J., Huska, D., Adam, V., Kizek, R., Hubalek, J., 2010. Magnetic nanoparticles and targeted drug delivering. Pharmacol. Res. 62, 144–149. Choy, J.H., Jung, J.S., Oh, J.M., Park, M., Jeong, J., Kang, Y.K., Han, O.J., 2004. Layered double hydroxide as an efficient drug reservoir for folate derivatives. Biomaterials 25, 3059–3064. Choy, J.H., Kwak, S.Y., Park, J.S., Jeong, Y.J., Portier, J., 1999. Intercalative nanohybrids of nucleoside monophosphates and DNA in layered metal hydroxide. J. Am. Chem. Soc. 121, 1399–1400. Darder, M., Aranda, P., Ruiz-Hitzky, E., 2007. Bionanocomposites: a new concept of ecological, bioinspired, and functional hybrid materials. Adv. Mater. 19, 1309–1319. Faccio, R., Pardo, H., Denis, P.A., Oeiras, R.Y., Araujo-Moreira, F.M., Verissimo-Alves, M., Mombrú, A.W., 2008. Magnetism induced by single carbon vacancies in a three-dimensional graphitic network. Phys. Rev. B 77.
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