Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 77–83
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Modification of chitosan by using samarium for potential use in drug delivery system Eny Kusrini a,⇑, Rita Arbianti a, Nofrijon Sofyan b, Mohd Aidil A. Abdullah c, Fika Andriani a a
Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus Baru UI, 16424 Depok, Indonesia Department of Metallurgical and Materials Engineering, Faculty of Engineering, Universitas Indonesia, Kampus Baru UI, 16424 Depok, Indonesia c Department of Chemical Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, Malaysia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Chitosan–Sm complexes were
synthesized by the impregnation method. 3+ Chitosan combined with Sm ions produced a drug carrier material with fluorescence properties. 3+ The addition of Sm ions into chitosan affects its physical and chemical properties. 3+ The Sm ion is used as an indicator of drug release with ibuprofen as a model drug. Chitosan–Sm 25 wt.% showed the highest efficiency of ibuprofen adsorption (33.04%).
a r t i c l e
i n f o
Article history: Received 15 November 2012 Received in revised form 16 September 2013 Accepted 29 September 2013 Available online 7 October 2013 Keywords: Chitosan Drug delivery Ibuprofen Fluorescence Samarium
a b s t r a c t In the presence of hydroxyl and amine groups, chitosan is highly reactive; therefore, it could be used as a carrier in drug delivery. For this study, chitosan–Sm complexes with different concentrations of samarium from 2.5 to 25 wt.% have been successfully synthesized by the impregnation method. Chitosan combined with Sm3+ ions produced a drug carrier material with fluorescence properties; thus, it could also be used as an indicator of drug release with ibuprofen (IBU) as a model drug. We evaluated the spectroscopic and interaction properties of chitosan and Sm3+ ions, the interaction of chitosan–Sm matrices with IBU as a model drug, and the effect of Sm3+ ions addition on the chitosan ability to adsorb the drug. The result showed that the hypersensitive fluorescence intensity of chitosan–Sm (2.5 wt.%) is higher than the others, even though the adsorption efficiency of chitosan–Sm 2.5 wt.% is lower (29.75%) than that of chitosan–Sm 25 wt.% (33.04%). Chitosan–Sm 25 wt.% showed the highest efficiency of adsorption of ibuprofen (33.04%). In the release process of ibuprofen from the chitosan–Sm–IBU matrix, the intensity of orange fluorescent properties in the hypersensitive peak of 4G5/2 ? 6H7/2 transition at 590 nm was observed. Fluorescent intensity increased with the cumulative amount of IBU released; therefore, the release of IBU from the Sm-modified chitosan complex can be monitored by the changes in fluorescent intensity. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Indonesia produces large amount of biological wastes, including shrimp waste, crab shells, and ox bones. Shrimp waste is promising ⇑ Corresponding author. Tel.: +62 21 7863516x6207; fax: +62 21 7863515. E-mail address: [email protected] (E. Kusrini). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.132
material having a high sales value because it contains protein, carotenoids, and chitin [1]. Chitin compounds in biological waste are part of a class of polysaccharides that can be converted to chitosan by deacetylation. Chitosan shows excellent potential as a biomaterial because of its biocompatibility in the mammalian body; it is a polymer biomaterial that is biodegradable and nontoxic to mammalian cells [2]. Due to these properties, therefore,
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it has the possibility to be used as a potential drug carrier in the drug delivery process. As such, the carrier material is used to modify the drug release profile, drug adsorption, and drug distribution in the body so that the medications would work optimally. In addition, the drug carrier is also used to encapsulate the drug to be released so that the drug is active only in targeted areas in the body. In this instance, as a drug carrier, chitosan is expected to encapsulate the drug so that the drug will be released only in accordance with the target disease in the mammalian body. Use of chitosan as a drug carrier has been widely reported [3– 10]. The cationic character in the amine groups of chitosan has responsibility in its application as drug delivery system [3]. Chitosan has higher nitrogen content compared to that of chitin, thus chitosan would be a better chelating agent compared to chitin [4]. Chitosan can be modified by combining it with other chemicals, such as N-Succinyl, glutaraldehyde, and glycyrrhetinic acid, for use as drug carriers for certain diseases [5–10]. Additionally, the use of lanthanide ions in drug carriers has also been widely studied [11–13]. These lanthanide ions emit fluorescent light caused by excitation of electrons. Emission color, referred to as fluorescence, can be used as an indicator of drug release in drug carrier systems. In the drug delivery system, lanthanide ions can function as sensors; their characteristic fluorescent intensity change could be used to identify drug release in the drug delivery process. When ibuprofen (IBU) is dissolved in aqueous solution, it will form the carboxylic group having a negative charge, whereas chitosan will have a positive charged. Thus, it is expected that IBU and chitosan will interact through an electrostatic bonding and or hydrogen bonding [14]. IBU is frequently used as a model drug for the purpose of sustained, controlled drug delivery and controlled release. This would enable straightforward measurements of release times, primarily because the IBU possess short biological half-life (2 h), conducive to pharmacological activity and has suitable molecule size (1.0–0.6 nm) [12]. However, IBU is an antisteroidal antiflammatory drug and has an amphiphilic property that may lead to stomach injury and or gastric irritation. Hence, encapsulation of the IBU by using chitosan or modified chitosan would reduce disorder effects and painful condition, especially to minimize the undesirable effects and prolong its anti-inflammatory character. In this study, chitosan was combined with samarium ion (Sm3+); lanthanide-type ion that emits orange fluorescent light with the transition region 4G5/2 ? 6H7/2 at a wavelength of 590 nm. Introducing of samarium (Sm) in chitosan was expected to increase chitosan ability to adsorb IBU as model drug. Based on the FTIR, fluorescence spectrophotometry, UV–Vis and SEM– EDX characterizations, we would have the idea about the interaction, morphological change and performance of the Sm-modified chitosan used in drug delivery system. Therefore, contributions of this study would be the use of chitosan as carriers in the drug delivery systems that is natural, nontoxic, biodegradable, and safe. Drug release from chitosan–Sm in dissolution media can be monitored by changes in the fluorescence of Sm3+ ion contained therein.
Materials and method Materials Chitosan medical grade powder with deacetylation degree of 90.77%, off white, viscosity of 18 cps, moisture content of 6.61%, ash content of 0.73%, protein content of 60.5%, pH 7–8 and molecular weight from 20,000 to 300,000 Mw was purchased from PT Biotech Surindo (West Java, Indonesia). Sm(NO3)36H2O was purchased from Sigma Aldrich (Wisconsin, USA). Distilled water,
ethanol, methanol, KH2PO4, NaOH, HCl, and lactic acid were purchased from PT Merck Tbk Indonesia. All materials in the study were used without any further purification. Synthesis of chitosan–Sm The chitosan–Sm was synthesized by impregnation method [15]. The Sm(NO3)36H2O with weight variations of 0.05, 0.1, 0.2, 0.3 and 0.5 g was dissolved in 100 mL of distilled water and added to chitosan (2 g). The solution was stirred with a magnetic stirrer at 500 rpm for 6 h and was filtered with a vacuum filter. The residue formed was washed with distilled water and was dried in an oven at 60 °C for 4 h. The resulting chitosan–Sm complex was crushed and weighed according to the mass. The yields of chitosan–Sm with Sm loading (2.5, 5, 10, 15, 25 wt.%) were 1.38, 1.21, 1.67, 1.94 and 2.05 g, respectively. Synthesis of chitosan–Sm–IBU The chitosan–Sm–IBU matrices were prepared in accordance with literature [13]. Each of chitosan–Sm (0.4 g) was added into 50 mL of ethanol containing IBU of 3 g. The mixture was stirred with a magnetic stirrer for 24 h before subsequently separated by centrifugation and the obtained precipitate was dried in an oven at 60 °C for 12 h. The resulting chitosan–Sm–IBU was crushed and weighed according to mass. The yields of chitosan–Sm–IBU with Sm loading from 2.5 to 25 wt.% were 0.4002, 0.4005, 0.4167, 0.4155 and 0.42 g, respectively. Preparation of drug adsorption and release system The experiments were carried out in a beaker containing methanol (25 mL) by mixing a 25 mg of chitosan–Sm–IBU with concentration variations of Sm loading from 2.5 to 25 wt.%. The solution was stirred and allowed to still for 24 h at room temperature. Each of sample solutions was then filtered, and IBU content was measured by using UV–Vis spectrophotometer. The drug release system was prepared by adding 10 mg of chitosan–Sm–IBU to five variations of the Sm loading in 50 mL of phosphate buffer (pH 7.4) in a sealed container. All the drug release systems of chitosan–Sm–IBU were stored in an incubator at 37 °C. Sampling of the systems was performed by taking 5 mL from each of the systems once per hour for 24 h. The UV–Vis adsorption spectral values were measured on a UV–Vis-spectrophotometer. The drug IBU adsorbed and released in vitro was performed in triplicate in order to obtain an accurate value during measurement. The fluorescence properties of all the samples were performed by using a spectrophotometer. For this measurement, the chitosan–Sm and chitosan–Sm–IBU were firstly dissolved in a lactic acid solution 5%. Physical measurements FTIR spectra were recorded on a Perkin–Elmer system 2000 FTIR spectrophotometer in the range of 4000–400 cm 1 by using the conventional KBr pellet method for solid samples. SEM–EDX measurements were performed by using the JEOL JSM-6360LA electron microscope at 20 kV and 30 mA. The loading amount of IBU in the materials was performed using the UV–Vis spectrophotometer (HITACHI U-2810) with a wavelength of 280 nm. Fluorescence properties of synthesized materials were characterized by using the HITACHI F-2000 fluorescence spectrophotometer with an excitation wavelength of 295 nm and an emission wavelength of 594 nm. All characterizations were carried out at room temperature.
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Results and discussion Characterization of materials FTIR studies of chitosan, chitosan–Sm, and chitosan–Sm–IBU Functional groups usually contain multiple bonds or lone pairs of electrons that make them very reactive. As has been stated earlier, the amine and hydroxyl groups of chitosan are very reactive to coordinate with the metallic ion, such as lanthanide ion. The FTIR spectra for chitosan, IBU, chitosan–Sm and chitosan– Sm–IBU are displayed in Figs. 1 and 2. The characteristic absorption band at 3383 cm 1 is assigned to the stretching vibration of the N–H group bonded to the O–H group, and the peaks at 1659 and 1600 cm 1 are attributed to the bands of amide I and II. The absorption band of N–H at 1600 cm 1 is more intense compared to the band at 1639 cm 1, indicating that the deacetylation process is more effective [16]. Since the deacetylation occurred, the absorption band at 1659 cm 1 decreases, whereas the absorption band at 1600 cm 1 increases, indicating the presence of amine group in chitosan [4]. In our study, the absorption band at 1600 cm 1 in chitosan is shifted to 1594–1597 cm 1, indicating coordination of Sm3+ with amine group. Both the amine and hydroxyl functional groups in chitosan are the most reactive and can bind Sm3+ ions. Bonding between the hydroxyl group and the Sm3+ ions leads to frequency changes from 3383 cm 1 (free chitosan) to 3388, 3379, 3377, 3373 and 3380 cm 1, for the chitosan–Sm matrices from 2.5 to 25 wt.%, respectively. A new absorption band at 1619– 1631 cm 1 is observed. This band is expected to be the bonding between chitosan and samarium ions. The amine or acetamide groups at C2 in chitosan are also associated with the samarium ions. The interaction between chitosan and samarium ion is shown in Fig. 3A. Indication of the adsorption of IBU onto the surface of chitosan– Sm was characterized by the appearance of carboxyl groups on the chitosan–Sm–IBU for five variations of Sm3+ ion concentrations, with a slight decrease in transmittance percentage. The band assigned to the vibration of –COOH at 1714 cm 1 is clearly observed in the IBU spectrum. It is also observed that the intensity of band absorption of –COOH group for the chitosan–Sm–IBU is decreased and shifted to a higher wavenumber (1717–1722 cm 1). The absorption bands of quaternary carbon atom at 1463 and 1508 cm 1, tertiary carbon atom at 1322 cm 1, hydroxyl bending vibration at 1420 cm 1 and C–H band at 2956 and 2925 cm 1 are observed in the IBU spectrum. In all of the chitosan–Sm–IBU spectra displayed the quaternary carbon atom, tertiary carbon atom,
Fig. 1. FTIR spectra of chitosan and chitosan–Sm, where a = chitosan, b = chitosan– Sm 2.5 wt.%, c = chitosan–Sm 5 wt.%, d = chitosan–Sm 10 wt.%, e = chitosan–Sm 15 wt.%, f = chitosan–Sm 25 wt.%.
Fig. 2. FTIR spectra of chitosan, IBU and chitosan–Sm–IBU, where a = chitosan, b = ibuprofen, c = chitosan–Sm–IBU 2.5 wt.%, d = chitosan–Sm–IBU 5 wt.%, e = chitosan–Sm–IBU 10 wt.%, f = chitosan–Sm–IBU 15 wt.%, g = chitosan–Sm–IBU 25 wt.%.
hydroxyl bending vibration, and C–H band are similar with the absorption peaks observed in the IBU. Thus, it is confirmed that the IBU is adsorbed onto the surface of chitosan–Sm. The OH groups in chitosan are important for bonding drug molecules [13]. These groups cause the formation of hydrogen bonds so that the absorption peaks appeared at 2951–2957 cm 1. The intensity of IBU molecule aromatic ring is observed in all of the chitosan– Sm–IBU, which is decreased and signified by the peak appearing at 780 cm 1. New absorption peak at about of 964–967 cm 1 is also observed. The vibration of Sm–O bond at 466–468 cm 1 is observed for all of chitosan–Sm–IBU spectra. The absorption peak of the secondary hydroxyl group in chitosan (1032 cm 1) and chitosan–Sm (1033 cm 1) is quite similar. However, in all of the chitosan–Sm–IBU spectra, this peak is shifted to 1021–1022 cm 1. It is confirmed that the IBU drug is bonded with chitosan through hydroxyl group of chitosan. The bands at 2890 and 1383 cm 1 in chitosan are assigned to the C–H stretching vibration in polymeric backbone and C–H bending, respectively. These peaks in all of chitosan–Sm spectra are slightly shifted to 2886–2894 cm 1 and 1383–1385 cm 1. The band at 1428 cm 1 in chitosan is attributed to the stretching vibration of C–N group. This peak is shifted to lower frequencies at 1424–1425 cm 1 for all the chitosan–Sm. Thus, the addition of Sm3+ ions into chitosan also affects the absorption peaks of the other functional groups present in the chitosan, namely C–H, C– O, and C–N. Upon complexation between Sm3+ and chitosan, the absorption peaks of functional groups in chitosan are shifted to lower frequencies. It could be argued that the addition of Sm3+ ions into chitosan affects its physical and chemical properties. Morphology and composition of chitosan, chitosan–Sm, and chitosan– Sm–IBU The morphologies of chitosan, chitosan–Sm, and chitosan–Sm– IBU were characterized by using SEM (Fig. S1a–c). SEM of pure chitosan reveals a flat structure and porous rods morphology. In Fig. S1b, chitosan–Sm appears rougher than that of pure chitosan and some porous are covered by the samarium metal ions. With the addition of IBU into chitosan–Sm, the morphology of chitosan–Sm–IBU becomes dense and more rugged than that of chitosan–Sm because a number of IBU on the surface of chitosan form a clot as can be seen in Fig. S1c. The SEM micrograph of chitosan–Sm–IBU also reveals that the samarium metal ions and IBU uniformly spread over the surface of chitosan forming rugged microstructure, indicating the presence of adsorbed ibuprofen molecules on the surface of chitosan. The ibuprofen drug is more
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Fig. 3. Possible molecule structure of chitosan and Sm (A) and possible interaction between IBU and chitosan (B). The dashed line (—) shows the hydrogen bonding.
Table 1 Efficiency adsorption of ibuprofen by the chitosan and chitosan–Sm. Sm loading (wt/wt.%)
Sample
Concentration of IBU (mg/mL)
Efficiency adsorption (%)
0 2.5 5 10 15 25
Chitosan–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU
15.97 17.85 19.51 19.75 19.82 19.82
26.61 29.75 32.51 32.92 33.03 33.04
efficiently encapsulated in the chitosan–Sm matrices. In further study, to evaluate the loading characteristic, drug of 60 mg/mL was loaded into chitosan–Sm with various concentration (2.5– 25 wt.%). The results show that the encapsulation efficiency of drug in the chitosan–Sm matrices increases with increasing concentration of samarium (Table 1). Identification of chemical contents in pure chitosan, selected samples of chitosan–Sm 25 wt.%, and chitosan–Sm–IBU was performed with by using EDX. Images of the EDX results are shown in Fig. S2a–c. The selected results of EDX for the chitosan–Sm 25 wt.% confirm the presence of Sm 3+ ions in
chitosan. Based on the EDX result, the mass of Sm3+ ions on the surface of chitosan is 2.75%, whereas the Sm3+ ion attached onto the surface of chitosan–Sm–IBU is 1.31%. This number decrease when compared with the Sm3+ ions in chitosan–Sm material. This decrease is expected to be due to the replacement of the samarium metal ions by the IBU molecules. IBU is adsorbed onto the surface of chitosan by impregnation method (Fig. S2a–c). The hydroxyl groups on the surface of chitosan would act as the reaction sites and form hydrogen bonding with the carboxyl group of IBU (Fig. 3B). Possible molecule structure interaction between the Sm-modified chitosan and IBU as model drug is illustrated in Fig. 4. Fluorescence properties of chitosan, chitosan–Sm, and chitosan–Sm– IBU Fluorescence properties of chitosan–Sm and chitosan–Sm–IBU were measured by using a fluorescence spectrophotometer. In the process of measurement, the emission wavelength was determined according to the characteristics of the Sm3+ ions. Fluorescence spectra for the chitosan–Sm and chitosan–Sm–IBU can be seen in Figs. 5 and 6. In all of the chitosan–Sm and chitosan– Sm–IBU samples, the hypersensitivity peak in the 4G5/2 ? 6H7/2
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Fig. 6. Fluoresence spectra of chitosan–Sm–IBU matrices at different concentration of Sm3+ ion, where a = chitosan–Sm 2.5 wt.%, b = chitosan–Sm 5 wt.%, c = chitosan– Sm 10 wt.%, d = chitosan–Sm 15 wt.%, e = chitosan–Sm 25 wt.%.
Drug adsorption and release system Fig. 4. Possible molecule structure and interaction between chitosan–Sm and IBU. The dashed line (—) shows the hydrogen bonding between the hydroxyl group of chitosan and carboxyl group of IBU molecules.
Fig. 5. Fluoresence spectra of the modified of chitosan–Sm with different concentration of Sm3+ ion, where a = chitosan–Sm 2.5 wt.%, b = chitosan–Sm 5 wt.%, c = chitosan–Sm 10 wt.%, d = chitosan–Sm 15 wt.%, e = chitosan–Sm 25 wt.%.
transition is contained in the emission wavelength of 590 nm. This finding is consistent with that proposed by Bünzli and Piguet [17] in which orange luminescence properties of Sm3+ ions can be seen in the transition region 4G5/2 ? 6H7/2 at an emission wavelength of 590 nm. Based on Figs. 5 and 6, it is observed that the emission intensity of the chitosan–Sm changes with the Sm3+ ion concentration. This change is expected to be due to the difference in energy transfer occurred in Sm3+ ions in the matrix. The lowest concentration of Sm loading in chitosan–Sm (2.5 wt.%) has the highest hypersensitive fluorescence intensity (4930 a.u.). The highest concentration of Sm loading in chitosan–Sm (25 wt.%) exhibits the hypersensitive fluorescence intensity at 4130 a.u, whereas the rest show hypersensitive fluorescence intensity from 3618 to 3970 a.u. These fluorescence properties of chitosan–Sm with various Sm loading could be used as an indication of its potential for applications in drug delivery/carrier systems. Relationship between the drug adsorption and release of IBU as a model drug in the chitosan–Sm will be discussed in the following section.
For this application, the drug IBU loading matrix was prepared by impregnation method to accommodate the drug IBU inside the Sm-modified chitosan. During the process, the IBU molecules will be released through the diffusion mechanism. The hydroxyl groups from the chitosan will form hydrogen bonding with the carboxyl group of IBU molecules since the IBU is adsorbed onto the surface of matrices. In the release process, the solvent enters the IBU molecules via their pores. The IBU drug is then slowly dissolved into the buffer solution from the surface and diffuses from the system. Adsorption of IBU molecules onto the Sm-modified chitosan complexes was performed by the incubation method. For this study, the addition of IBU molecules into the chitosan–Sm material was carried out in accordance with the literature [10]. We noted that selected drug IBU would physically stick to the Sm-modified chitosan or be adsorbed onto the surface of chitosan–Sm [18]. In addition, interaction of the IBU molecules with the chitosan–Sm complex can also take place by hydrogen bonding between the OH group from chitosan and carboxylic groups from IBU molecules when the IBU is adsorbed onto the surface of chitosan (Fig. 4). During the process, chitosan–Sm complex will encapsulate the IBU molecules so that the rate of diffusion of IBU molecules can be controlled and monitored. Determination of IBU content in the chitosan–Sm by UV–Vis spectrophotometer was conducted to determine the adsorption efficiency of IBU molecules in the chitosan–Sm complex and the effect of Sm3+ ions addition on chitosan adsorption of IBU molecules. The adsorption efficiency of IBU molecules in the chitosan–Sm complex is greater than the adsorption of IBU molecules by pure chitosan (Table 1). The adsorption of IBU molecules by the Sm-free chitosan is about 26.61%. Adsorption efficiency increases with increasing concentrations of Sm3+ ions contained in the chitosan. These results indicate that Sm3+ ions affect chitosan ability to adsorb IBU molecules. Chitosan–Sm–IBU with the Sm loading of 25 wt.% has adsorption efficiency of IBU molecules of 33.04%. This amount is almost the same with the adsorption efficiency of IBU (33.03%) by the chitosan–Sm–IBU with Sm3+ loading of 15 wt.%. This signifies that the Sm3+ loading in chitosan in the range of15–25 wt.% is optimum for the IBU adsorption onto the chitosan–Sm matrices. During adsorption of IBU molecules onto pure chitosan and chitosan– Sm, the carboxyl groups contained in the IBU form bonds with
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the hydroxyl groups from chitosan (Figs. 3B and 4). The addition of Sm3+ ions into chitosan will create bonding between the ions and the hydroxyl and amine groups of the chitosan (Fig. 3A). This will then increase the bond strength in these groups and thus they would become more reactive to bind the drug molecules. In addition, the modification of chitosan with the addition of Sm3+ ions will increase its reactivity, as noted by the increase in adsorption efficiency of IBU with the increasing content of Sm3+ ions in the chitosan. Determination of the IBU release profile of chitosan–Sm–IBU is necessary to understand the difference in profiles of pure chitosan with chitosan–Sm with various Sm loading. In addition, determination of the IBU release profile of the chitosan–Sm–IBU complex was performed to understand the influence of the presence of Sm3+ ions on the rate of IBU release from chitosan–Sm–IBU matrices. IBU release profiles from the chitosan–Sm–IBU matrices are shown in Fig. 7. The IBU adsorption pattern was conducted in triplicate. Standard deviation for each measurement in various Sm loading from 0 to 25 wt.% is 0.00043, 0.00099, 0.02607, 0.00073, 0.00171 and 0.00037, respectively. The in vitro release tests of IBU in the chitosan–Sm–IBU matrices were performed at pH 7.4, which is the condition in the intestinal fluid, for 24 h. In general, the drug release from chitosan occurs through a diffusion mechanism that swells the chitosan matrix [18]. In Fig. 7, it can be seen that the drug release profiles for pure chitosan and chitosan–Sm–IBU with five concentration variations of Sm3+ ions are quite similar. For all the drug release profiles, IBU is released slowly, reaching 50% in 8 hand perfectly separating in 24 h. Based on the graph in Fig. 7, it can be observed that the release mechanism of IBU from pure chitosan and chitosan–Sm–IBU depends on the diffusion mechanism. In this process, IBU molecules diffused slowly from pure chitosan and chitosan–Sm–IBU matrices. The drug release of IBU from both matrices begins with the swelling of chitosan–Sm as a drug coating material, followed by a slow diffusion of IBU molecules into the dissolution medium of the phosphate buffer pH 7.4 [13]. IBU diffusion is possible because of differences in the concentrations of IBU in the dissolution media of phosphate buffer pH 7.4 and the IBU in the chitosan–Sm. The slow rate of drug release of IBU could due to the strong hydrogen bonding and interaction between IBU molecules and the functional groups of the chitosan. Based on the graph, it can also be observed that the release of IBU from the chitosan–Sm–IBU matrices is slower than the release of IBU from pure chitosan. This could be due to the presence of Sm3+ ions that increase the reactivity of
Fig. 7. Drug release profile of ibuprofen in the chitosan–Sm–IBU complex, where 0 = chitosan, a = chitosan–Sm–IBU 2.5 wt.%, b = chitosan–Sm–IBU 5 wt.%, c = chitosan–Sm–IBU 10 wt.%, d = chitosan–Sm–IBU 15 wt.%, e = chitosan–Sm–IBU 25 wt.%. Standard deviation with confidence level of 95.0% for each measurement in various Sm loading from 0 to 25 wt.% are 0.00043, 0.00099, 0.02607, 0.00073, 0.00171 and 0.00037, respectively.
hydroxyl groups of chitosan, thus producing a strong hydrogen bond between hydroxyl groups from chitosan and the carboxyl group of IBU. For all variations, the drug release profile continues to rise to reach an equilibrium point after 12 h. This finding is consistent with the human digestive system time of 8 h. IBU released from the chitosan–Sm–IBU matrices in the phosphate buffer at pH 7.4 enters the matrices of pure chitosan and chitosan–Sm–IBU. These matrices expand and swell causing IBU molecules diffuse into the phosphate buffer until the concentration of IBU in the phosphate buffer is the same as the concentration of IBU in the pure chitosan and chitosan–Sm–IBU matrices at the time of synthesis. The addition of Sm3+ ions into chitosan was intended to allow the drug release from chitosan–Sm–IBU could be monitored through the changes in fluorescent intensity. The relationship between these intensity changes and the cumulative amount of IBU released from the chitosan–Sm–IBU matrices is shown in Fig. 8. Fluorescent intensity of the drug release samples was measured according to the characteristics of Sm3+ ions, which emit fluorescence with the 4G5/2 ? 6H7/2 transition at 590 nm and the excitation wavelength of 295 nm. Based on Fig. 8, it can be seen that the fluorescent intensity for all variations of the chitosan–Sm– IBU matrices increases with the cumulative amount of IBU released from the chitosan–Sm–IBU matrices. This finding is similar to the results reported by Yang et al. [13]. When IBU molecules are adsorbed onto the surface of chitosan, hydrogen bonding occurs between the hydroxyl group of chitosan and the carboxyl group of IBU causes the Sm3+ ions bonding with the hydroxyl groups in chitosan to weaken [13]. Therefore, fluorescent intensity of the Sm3+ ions in the matrices of chitosan–Sm–IBU will be weakened. In the process of drug release, IBU slowly detaches from the matrices indicated by weakening of the bond between chitosan hydroxyl group and IBU carboxyl group. At the same time, the bond between the Sm3+ions and hydroxyl groups in chitosan will rebound. This will cause the fluorescent intensity to increase with the cumulative amount of IBU released from the chitosan–Sm–IBU matrices. At the beginning, the cumulative release pattern of IBU (below 40%) from the chitosan–Sm are similar. Later on, the pattern changes after the cumulative release of IBU is greater than 45%. This may be due to the similar surface as well as interaction between IBU and chitosan and/or chitosan–Sm. The Sm-modified chitosan (2.5 wt.%) gives the highest intensity compared to the others chitosan–Sm (Fig. 8). The cumulative release of IBU (5%) from chitosan–Sm (2.5 wt.%) occurs at a fluorescence intensity of 4221 a.u., then slightly increases and reaches 4366 a.u. for the cumulative release of IBU (38.7%). Furthermore, increasing the cumulative release of IBU until 90.8% take place at a fluorescence
Fig. 8. Comparison of fluorescence intensity for drug release activity, where a = chitosan–Sm–IBU 2.5 wt.%, b = chitosan–Sm–IBU 5 wt.%, c = chitosan–Sm–IBU 10 wt.%, d = chitosan–Sm–IBU 15 wt.%, e = chitosan–Sm–IBU 25 wt.%.
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intensity of 6431 a.u. On the other hand, the cumulative release of IBU from chitosan–Sm 25 wt.% gradually increases and reaches 47.3% at a fluorescence intensity of 2952 a.u. After that, the cumulative release of IBU is faster and sharper compared to that of the others of matrices, even though it still occurs below of that the chitosan–Sm 2.5 wt.%. Finally, the cumulative release of IBU (100%) takes place at fluorescence intensities of 6653; 5701; 4857; 5261 and 6100 a.u., respectively for the chitosan–Sm (2.5– 25 wt.%) (see again Fig. 8). It is found that the lowest concentration of Sm loading in chitosan–Sm (2.5 wt.%) has the hypersensitive fluorescence intensity higher than the others, even though the adsorption efficiency of chitosan–Sm 2.5 wt.% is lower (29.75%) compared to the chitosan–Sm 25 wt.% (33.04%). Conclusion The chitosan–Sm complexes with different concentrations of Sm from 0 to 25 wt.% have been successfully synthesized by the impregnation method. The concentration variation of Sm loading in chitosan was introduced to understand the influence of Sm3+ ion content on chitosan ability to adsorb a drug and the drug release process. The addition of Sm3+ ions into chitosan enhances its ability to adsorb IBU molecules as a model drug. The results show that the hypersensitive fluorescence intensity of chitosan– Sm (2.5 wt.%) is higher than the others, even though its adsorption efficiency is lower (29.75%) compared to that of chitosan–Sm 25 wt.% (33.04%). Chitosan and Sm could be used to modify a drug release profile, drug adsorption, and drug distribution in the body so that medications will work optimally and can be monitored. Fluorescence properties of the Sm3+ ions could be used to indicate IBU release from the chitosan–Sm–IBU matrices; the change in fluorescent intensity takes place in the hypersensitive peak at 590 nm. Chitosan–Sm 25 wt.% demonstrated the highest adsorption efficiency of IBU (33.04%). Based on bioactivity of chitosan and fluorescent properties of Sm3+ ions, chitosan–Sm complexes would be promising for potential applications in the field of drug delivery and disease therapy.
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Acknowledgements This study was supported by Hibah Fundamental DIKTI, Ministry of Education and Culture Republic of Indonesia. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.saa.2013.09.132. References [1] P. Lertsutthiwong, N.C. How, S. Chandrkrachang, W Stevens, J. Met. Mater. Miner. 12 (2002) 11–18. [2] D.W. Lee, H. Lim, H.N. Chong, W.S. Shim, Open Biomater. J. 1 (2009) 10–20. [3] A. Bernkop-Schnürch, S. Dünnhaupt, Euro. J. Pharm. Biopharm. 81 (2012) 463– 469. [4] A. Bansiwal, D. Thakrea, N. Labhshetwara, S. Meshram, S. Rayalu, Colloids Surf. B: Biointerf. 74 (2009) 216–224. [5] S. Senel, M.J. Kremer, S. Kas, P.W. Wertz, A.A. Hincal, C.A. Squier, Biomaterials 21 (2000) 2067–2071. [6] Y. Kato, H. Onishi, Y. Machida, Biomaterials 25 (2004) 907–915. [7] P. Chan, M. Kurisawa, J.E. Chung, Y.Y. Yang, Biomaterials 28 (2007) 540–549. [8] H. Wang, P. Zhao, X. Liang, X. Gong, T. Song, R. Niu, J. Chang, Biomaterials 31 (2010) 4129–4138. [9] Q. Tian, C.N. Zhang, X.H. Wang, W. Wang, W. Huang, R.T. Cha, C.H. Wang, Z. Yuan, M. Liu, H.Y. Wan, H. Tang, Biomaterials 31 (2010) 4748–4756. [10] S.R. Jameela, A. Jayakrishnan, Biomaterials 16 (1995) 769–775. [11] Y. Fan, P. Yang, S. Huang, J. Jiang, H. Lian, J. Lin, J. Phys. Chem. C113 (2009) 7826–7830. [12] P. Yang, Z. Quan, C. Li, X. Kang, H. Lian, J Lin, Biomaterials 29 (2008) 4341– 4347. [13] S. Setua, D. Menon, A. Asok, S. Nair, M. Koyakutty, Biomaterials 31 (2010) 714– 729. [14] I.A. Sogias, A.C. Williams, V.V. Khutoryanskiy, Int. J. Pharm. 436 (2012) 602– 610. [15] R. Yao, F. Meng, L. Zhang, D. Ma, M. Wang, J. Hazard. Mat. 165 (2009) 454–460. [16] A.T. Pauline, J.I. Simionato, J.C. Garcia, J. Nozaki, Carbohydr. Polym. 64 (2006) 98–103. [17] J.G. Bünzli, C. Piguet, Chem. Soc. Rev. 34 (2005) 1048–1077. [18] S.A. Agnihotri, N.N. Mallikarjuna, T.M. Aminabhavi, J. Controlled Release 100 (2004) 5–28.
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Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy journal homepage: www.elsevier.com/locate/saa
Modification of chitosan by using samarium for potential use in drug delivery system Eny Kusrini a,⇑, Rita Arbianti a, Nofrijon Sofyan b, Mohd Aidil A. Abdullah c, Fika Andriani a a
Department of Chemical Engineering, Faculty of Engineering, Universitas Indonesia, Kampus Baru UI, 16424 Depok, Indonesia Department of Metallurgical and Materials Engineering, Faculty of Engineering, Universitas Indonesia, Kampus Baru UI, 16424 Depok, Indonesia c Department of Chemical Sciences, Faculty of Science and Technology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu Darul Iman, Malaysia b
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Chitosan–Sm complexes were
synthesized by the impregnation method. 3+ Chitosan combined with Sm ions produced a drug carrier material with fluorescence properties. 3+ The addition of Sm ions into chitosan affects its physical and chemical properties. 3+ The Sm ion is used as an indicator of drug release with ibuprofen as a model drug. Chitosan–Sm 25 wt.% showed the highest efficiency of ibuprofen adsorption (33.04%).
a r t i c l e
i n f o
Article history: Received 15 November 2012 Received in revised form 16 September 2013 Accepted 29 September 2013 Available online 7 October 2013 Keywords: Chitosan Drug delivery Ibuprofen Fluorescence Samarium
a b s t r a c t In the presence of hydroxyl and amine groups, chitosan is highly reactive; therefore, it could be used as a carrier in drug delivery. For this study, chitosan–Sm complexes with different concentrations of samarium from 2.5 to 25 wt.% have been successfully synthesized by the impregnation method. Chitosan combined with Sm3+ ions produced a drug carrier material with fluorescence properties; thus, it could also be used as an indicator of drug release with ibuprofen (IBU) as a model drug. We evaluated the spectroscopic and interaction properties of chitosan and Sm3+ ions, the interaction of chitosan–Sm matrices with IBU as a model drug, and the effect of Sm3+ ions addition on the chitosan ability to adsorb the drug. The result showed that the hypersensitive fluorescence intensity of chitosan–Sm (2.5 wt.%) is higher than the others, even though the adsorption efficiency of chitosan–Sm 2.5 wt.% is lower (29.75%) than that of chitosan–Sm 25 wt.% (33.04%). Chitosan–Sm 25 wt.% showed the highest efficiency of adsorption of ibuprofen (33.04%). In the release process of ibuprofen from the chitosan–Sm–IBU matrix, the intensity of orange fluorescent properties in the hypersensitive peak of 4G5/2 ? 6H7/2 transition at 590 nm was observed. Fluorescent intensity increased with the cumulative amount of IBU released; therefore, the release of IBU from the Sm-modified chitosan complex can be monitored by the changes in fluorescent intensity. Ó 2013 Elsevier B.V. All rights reserved.
Introduction Indonesia produces large amount of biological wastes, including shrimp waste, crab shells, and ox bones. Shrimp waste is promising ⇑ Corresponding author. Tel.: +62 21 7863516x6207; fax: +62 21 7863515. E-mail address: [email protected] (E. Kusrini). 1386-1425/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.saa.2013.09.132
material having a high sales value because it contains protein, carotenoids, and chitin [1]. Chitin compounds in biological waste are part of a class of polysaccharides that can be converted to chitosan by deacetylation. Chitosan shows excellent potential as a biomaterial because of its biocompatibility in the mammalian body; it is a polymer biomaterial that is biodegradable and nontoxic to mammalian cells [2]. Due to these properties, therefore,
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it has the possibility to be used as a potential drug carrier in the drug delivery process. As such, the carrier material is used to modify the drug release profile, drug adsorption, and drug distribution in the body so that the medications would work optimally. In addition, the drug carrier is also used to encapsulate the drug to be released so that the drug is active only in targeted areas in the body. In this instance, as a drug carrier, chitosan is expected to encapsulate the drug so that the drug will be released only in accordance with the target disease in the mammalian body. Use of chitosan as a drug carrier has been widely reported [3– 10]. The cationic character in the amine groups of chitosan has responsibility in its application as drug delivery system [3]. Chitosan has higher nitrogen content compared to that of chitin, thus chitosan would be a better chelating agent compared to chitin [4]. Chitosan can be modified by combining it with other chemicals, such as N-Succinyl, glutaraldehyde, and glycyrrhetinic acid, for use as drug carriers for certain diseases [5–10]. Additionally, the use of lanthanide ions in drug carriers has also been widely studied [11–13]. These lanthanide ions emit fluorescent light caused by excitation of electrons. Emission color, referred to as fluorescence, can be used as an indicator of drug release in drug carrier systems. In the drug delivery system, lanthanide ions can function as sensors; their characteristic fluorescent intensity change could be used to identify drug release in the drug delivery process. When ibuprofen (IBU) is dissolved in aqueous solution, it will form the carboxylic group having a negative charge, whereas chitosan will have a positive charged. Thus, it is expected that IBU and chitosan will interact through an electrostatic bonding and or hydrogen bonding [14]. IBU is frequently used as a model drug for the purpose of sustained, controlled drug delivery and controlled release. This would enable straightforward measurements of release times, primarily because the IBU possess short biological half-life (2 h), conducive to pharmacological activity and has suitable molecule size (1.0–0.6 nm) [12]. However, IBU is an antisteroidal antiflammatory drug and has an amphiphilic property that may lead to stomach injury and or gastric irritation. Hence, encapsulation of the IBU by using chitosan or modified chitosan would reduce disorder effects and painful condition, especially to minimize the undesirable effects and prolong its anti-inflammatory character. In this study, chitosan was combined with samarium ion (Sm3+); lanthanide-type ion that emits orange fluorescent light with the transition region 4G5/2 ? 6H7/2 at a wavelength of 590 nm. Introducing of samarium (Sm) in chitosan was expected to increase chitosan ability to adsorb IBU as model drug. Based on the FTIR, fluorescence spectrophotometry, UV–Vis and SEM– EDX characterizations, we would have the idea about the interaction, morphological change and performance of the Sm-modified chitosan used in drug delivery system. Therefore, contributions of this study would be the use of chitosan as carriers in the drug delivery systems that is natural, nontoxic, biodegradable, and safe. Drug release from chitosan–Sm in dissolution media can be monitored by changes in the fluorescence of Sm3+ ion contained therein.
Materials and method Materials Chitosan medical grade powder with deacetylation degree of 90.77%, off white, viscosity of 18 cps, moisture content of 6.61%, ash content of 0.73%, protein content of 60.5%, pH 7–8 and molecular weight from 20,000 to 300,000 Mw was purchased from PT Biotech Surindo (West Java, Indonesia). Sm(NO3)36H2O was purchased from Sigma Aldrich (Wisconsin, USA). Distilled water,
ethanol, methanol, KH2PO4, NaOH, HCl, and lactic acid were purchased from PT Merck Tbk Indonesia. All materials in the study were used without any further purification. Synthesis of chitosan–Sm The chitosan–Sm was synthesized by impregnation method [15]. The Sm(NO3)36H2O with weight variations of 0.05, 0.1, 0.2, 0.3 and 0.5 g was dissolved in 100 mL of distilled water and added to chitosan (2 g). The solution was stirred with a magnetic stirrer at 500 rpm for 6 h and was filtered with a vacuum filter. The residue formed was washed with distilled water and was dried in an oven at 60 °C for 4 h. The resulting chitosan–Sm complex was crushed and weighed according to the mass. The yields of chitosan–Sm with Sm loading (2.5, 5, 10, 15, 25 wt.%) were 1.38, 1.21, 1.67, 1.94 and 2.05 g, respectively. Synthesis of chitosan–Sm–IBU The chitosan–Sm–IBU matrices were prepared in accordance with literature [13]. Each of chitosan–Sm (0.4 g) was added into 50 mL of ethanol containing IBU of 3 g. The mixture was stirred with a magnetic stirrer for 24 h before subsequently separated by centrifugation and the obtained precipitate was dried in an oven at 60 °C for 12 h. The resulting chitosan–Sm–IBU was crushed and weighed according to mass. The yields of chitosan–Sm–IBU with Sm loading from 2.5 to 25 wt.% were 0.4002, 0.4005, 0.4167, 0.4155 and 0.42 g, respectively. Preparation of drug adsorption and release system The experiments were carried out in a beaker containing methanol (25 mL) by mixing a 25 mg of chitosan–Sm–IBU with concentration variations of Sm loading from 2.5 to 25 wt.%. The solution was stirred and allowed to still for 24 h at room temperature. Each of sample solutions was then filtered, and IBU content was measured by using UV–Vis spectrophotometer. The drug release system was prepared by adding 10 mg of chitosan–Sm–IBU to five variations of the Sm loading in 50 mL of phosphate buffer (pH 7.4) in a sealed container. All the drug release systems of chitosan–Sm–IBU were stored in an incubator at 37 °C. Sampling of the systems was performed by taking 5 mL from each of the systems once per hour for 24 h. The UV–Vis adsorption spectral values were measured on a UV–Vis-spectrophotometer. The drug IBU adsorbed and released in vitro was performed in triplicate in order to obtain an accurate value during measurement. The fluorescence properties of all the samples were performed by using a spectrophotometer. For this measurement, the chitosan–Sm and chitosan–Sm–IBU were firstly dissolved in a lactic acid solution 5%. Physical measurements FTIR spectra were recorded on a Perkin–Elmer system 2000 FTIR spectrophotometer in the range of 4000–400 cm 1 by using the conventional KBr pellet method for solid samples. SEM–EDX measurements were performed by using the JEOL JSM-6360LA electron microscope at 20 kV and 30 mA. The loading amount of IBU in the materials was performed using the UV–Vis spectrophotometer (HITACHI U-2810) with a wavelength of 280 nm. Fluorescence properties of synthesized materials were characterized by using the HITACHI F-2000 fluorescence spectrophotometer with an excitation wavelength of 295 nm and an emission wavelength of 594 nm. All characterizations were carried out at room temperature.
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Results and discussion Characterization of materials FTIR studies of chitosan, chitosan–Sm, and chitosan–Sm–IBU Functional groups usually contain multiple bonds or lone pairs of electrons that make them very reactive. As has been stated earlier, the amine and hydroxyl groups of chitosan are very reactive to coordinate with the metallic ion, such as lanthanide ion. The FTIR spectra for chitosan, IBU, chitosan–Sm and chitosan– Sm–IBU are displayed in Figs. 1 and 2. The characteristic absorption band at 3383 cm 1 is assigned to the stretching vibration of the N–H group bonded to the O–H group, and the peaks at 1659 and 1600 cm 1 are attributed to the bands of amide I and II. The absorption band of N–H at 1600 cm 1 is more intense compared to the band at 1639 cm 1, indicating that the deacetylation process is more effective [16]. Since the deacetylation occurred, the absorption band at 1659 cm 1 decreases, whereas the absorption band at 1600 cm 1 increases, indicating the presence of amine group in chitosan [4]. In our study, the absorption band at 1600 cm 1 in chitosan is shifted to 1594–1597 cm 1, indicating coordination of Sm3+ with amine group. Both the amine and hydroxyl functional groups in chitosan are the most reactive and can bind Sm3+ ions. Bonding between the hydroxyl group and the Sm3+ ions leads to frequency changes from 3383 cm 1 (free chitosan) to 3388, 3379, 3377, 3373 and 3380 cm 1, for the chitosan–Sm matrices from 2.5 to 25 wt.%, respectively. A new absorption band at 1619– 1631 cm 1 is observed. This band is expected to be the bonding between chitosan and samarium ions. The amine or acetamide groups at C2 in chitosan are also associated with the samarium ions. The interaction between chitosan and samarium ion is shown in Fig. 3A. Indication of the adsorption of IBU onto the surface of chitosan– Sm was characterized by the appearance of carboxyl groups on the chitosan–Sm–IBU for five variations of Sm3+ ion concentrations, with a slight decrease in transmittance percentage. The band assigned to the vibration of –COOH at 1714 cm 1 is clearly observed in the IBU spectrum. It is also observed that the intensity of band absorption of –COOH group for the chitosan–Sm–IBU is decreased and shifted to a higher wavenumber (1717–1722 cm 1). The absorption bands of quaternary carbon atom at 1463 and 1508 cm 1, tertiary carbon atom at 1322 cm 1, hydroxyl bending vibration at 1420 cm 1 and C–H band at 2956 and 2925 cm 1 are observed in the IBU spectrum. In all of the chitosan–Sm–IBU spectra displayed the quaternary carbon atom, tertiary carbon atom,
Fig. 1. FTIR spectra of chitosan and chitosan–Sm, where a = chitosan, b = chitosan– Sm 2.5 wt.%, c = chitosan–Sm 5 wt.%, d = chitosan–Sm 10 wt.%, e = chitosan–Sm 15 wt.%, f = chitosan–Sm 25 wt.%.
Fig. 2. FTIR spectra of chitosan, IBU and chitosan–Sm–IBU, where a = chitosan, b = ibuprofen, c = chitosan–Sm–IBU 2.5 wt.%, d = chitosan–Sm–IBU 5 wt.%, e = chitosan–Sm–IBU 10 wt.%, f = chitosan–Sm–IBU 15 wt.%, g = chitosan–Sm–IBU 25 wt.%.
hydroxyl bending vibration, and C–H band are similar with the absorption peaks observed in the IBU. Thus, it is confirmed that the IBU is adsorbed onto the surface of chitosan–Sm. The OH groups in chitosan are important for bonding drug molecules [13]. These groups cause the formation of hydrogen bonds so that the absorption peaks appeared at 2951–2957 cm 1. The intensity of IBU molecule aromatic ring is observed in all of the chitosan– Sm–IBU, which is decreased and signified by the peak appearing at 780 cm 1. New absorption peak at about of 964–967 cm 1 is also observed. The vibration of Sm–O bond at 466–468 cm 1 is observed for all of chitosan–Sm–IBU spectra. The absorption peak of the secondary hydroxyl group in chitosan (1032 cm 1) and chitosan–Sm (1033 cm 1) is quite similar. However, in all of the chitosan–Sm–IBU spectra, this peak is shifted to 1021–1022 cm 1. It is confirmed that the IBU drug is bonded with chitosan through hydroxyl group of chitosan. The bands at 2890 and 1383 cm 1 in chitosan are assigned to the C–H stretching vibration in polymeric backbone and C–H bending, respectively. These peaks in all of chitosan–Sm spectra are slightly shifted to 2886–2894 cm 1 and 1383–1385 cm 1. The band at 1428 cm 1 in chitosan is attributed to the stretching vibration of C–N group. This peak is shifted to lower frequencies at 1424–1425 cm 1 for all the chitosan–Sm. Thus, the addition of Sm3+ ions into chitosan also affects the absorption peaks of the other functional groups present in the chitosan, namely C–H, C– O, and C–N. Upon complexation between Sm3+ and chitosan, the absorption peaks of functional groups in chitosan are shifted to lower frequencies. It could be argued that the addition of Sm3+ ions into chitosan affects its physical and chemical properties. Morphology and composition of chitosan, chitosan–Sm, and chitosan– Sm–IBU The morphologies of chitosan, chitosan–Sm, and chitosan–Sm– IBU were characterized by using SEM (Fig. S1a–c). SEM of pure chitosan reveals a flat structure and porous rods morphology. In Fig. S1b, chitosan–Sm appears rougher than that of pure chitosan and some porous are covered by the samarium metal ions. With the addition of IBU into chitosan–Sm, the morphology of chitosan–Sm–IBU becomes dense and more rugged than that of chitosan–Sm because a number of IBU on the surface of chitosan form a clot as can be seen in Fig. S1c. The SEM micrograph of chitosan–Sm–IBU also reveals that the samarium metal ions and IBU uniformly spread over the surface of chitosan forming rugged microstructure, indicating the presence of adsorbed ibuprofen molecules on the surface of chitosan. The ibuprofen drug is more
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Fig. 3. Possible molecule structure of chitosan and Sm (A) and possible interaction between IBU and chitosan (B). The dashed line (—) shows the hydrogen bonding.
Table 1 Efficiency adsorption of ibuprofen by the chitosan and chitosan–Sm. Sm loading (wt/wt.%)
Sample
Concentration of IBU (mg/mL)
Efficiency adsorption (%)
0 2.5 5 10 15 25
Chitosan–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU Chitosan–Sm–IBU
15.97 17.85 19.51 19.75 19.82 19.82
26.61 29.75 32.51 32.92 33.03 33.04
efficiently encapsulated in the chitosan–Sm matrices. In further study, to evaluate the loading characteristic, drug of 60 mg/mL was loaded into chitosan–Sm with various concentration (2.5– 25 wt.%). The results show that the encapsulation efficiency of drug in the chitosan–Sm matrices increases with increasing concentration of samarium (Table 1). Identification of chemical contents in pure chitosan, selected samples of chitosan–Sm 25 wt.%, and chitosan–Sm–IBU was performed with by using EDX. Images of the EDX results are shown in Fig. S2a–c. The selected results of EDX for the chitosan–Sm 25 wt.% confirm the presence of Sm 3+ ions in
chitosan. Based on the EDX result, the mass of Sm3+ ions on the surface of chitosan is 2.75%, whereas the Sm3+ ion attached onto the surface of chitosan–Sm–IBU is 1.31%. This number decrease when compared with the Sm3+ ions in chitosan–Sm material. This decrease is expected to be due to the replacement of the samarium metal ions by the IBU molecules. IBU is adsorbed onto the surface of chitosan by impregnation method (Fig. S2a–c). The hydroxyl groups on the surface of chitosan would act as the reaction sites and form hydrogen bonding with the carboxyl group of IBU (Fig. 3B). Possible molecule structure interaction between the Sm-modified chitosan and IBU as model drug is illustrated in Fig. 4. Fluorescence properties of chitosan, chitosan–Sm, and chitosan–Sm– IBU Fluorescence properties of chitosan–Sm and chitosan–Sm–IBU were measured by using a fluorescence spectrophotometer. In the process of measurement, the emission wavelength was determined according to the characteristics of the Sm3+ ions. Fluorescence spectra for the chitosan–Sm and chitosan–Sm–IBU can be seen in Figs. 5 and 6. In all of the chitosan–Sm and chitosan– Sm–IBU samples, the hypersensitivity peak in the 4G5/2 ? 6H7/2
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Fig. 6. Fluoresence spectra of chitosan–Sm–IBU matrices at different concentration of Sm3+ ion, where a = chitosan–Sm 2.5 wt.%, b = chitosan–Sm 5 wt.%, c = chitosan– Sm 10 wt.%, d = chitosan–Sm 15 wt.%, e = chitosan–Sm 25 wt.%.
Drug adsorption and release system Fig. 4. Possible molecule structure and interaction between chitosan–Sm and IBU. The dashed line (—) shows the hydrogen bonding between the hydroxyl group of chitosan and carboxyl group of IBU molecules.
Fig. 5. Fluoresence spectra of the modified of chitosan–Sm with different concentration of Sm3+ ion, where a = chitosan–Sm 2.5 wt.%, b = chitosan–Sm 5 wt.%, c = chitosan–Sm 10 wt.%, d = chitosan–Sm 15 wt.%, e = chitosan–Sm 25 wt.%.
transition is contained in the emission wavelength of 590 nm. This finding is consistent with that proposed by Bünzli and Piguet [17] in which orange luminescence properties of Sm3+ ions can be seen in the transition region 4G5/2 ? 6H7/2 at an emission wavelength of 590 nm. Based on Figs. 5 and 6, it is observed that the emission intensity of the chitosan–Sm changes with the Sm3+ ion concentration. This change is expected to be due to the difference in energy transfer occurred in Sm3+ ions in the matrix. The lowest concentration of Sm loading in chitosan–Sm (2.5 wt.%) has the highest hypersensitive fluorescence intensity (4930 a.u.). The highest concentration of Sm loading in chitosan–Sm (25 wt.%) exhibits the hypersensitive fluorescence intensity at 4130 a.u, whereas the rest show hypersensitive fluorescence intensity from 3618 to 3970 a.u. These fluorescence properties of chitosan–Sm with various Sm loading could be used as an indication of its potential for applications in drug delivery/carrier systems. Relationship between the drug adsorption and release of IBU as a model drug in the chitosan–Sm will be discussed in the following section.
For this application, the drug IBU loading matrix was prepared by impregnation method to accommodate the drug IBU inside the Sm-modified chitosan. During the process, the IBU molecules will be released through the diffusion mechanism. The hydroxyl groups from the chitosan will form hydrogen bonding with the carboxyl group of IBU molecules since the IBU is adsorbed onto the surface of matrices. In the release process, the solvent enters the IBU molecules via their pores. The IBU drug is then slowly dissolved into the buffer solution from the surface and diffuses from the system. Adsorption of IBU molecules onto the Sm-modified chitosan complexes was performed by the incubation method. For this study, the addition of IBU molecules into the chitosan–Sm material was carried out in accordance with the literature [10]. We noted that selected drug IBU would physically stick to the Sm-modified chitosan or be adsorbed onto the surface of chitosan–Sm [18]. In addition, interaction of the IBU molecules with the chitosan–Sm complex can also take place by hydrogen bonding between the OH group from chitosan and carboxylic groups from IBU molecules when the IBU is adsorbed onto the surface of chitosan (Fig. 4). During the process, chitosan–Sm complex will encapsulate the IBU molecules so that the rate of diffusion of IBU molecules can be controlled and monitored. Determination of IBU content in the chitosan–Sm by UV–Vis spectrophotometer was conducted to determine the adsorption efficiency of IBU molecules in the chitosan–Sm complex and the effect of Sm3+ ions addition on chitosan adsorption of IBU molecules. The adsorption efficiency of IBU molecules in the chitosan–Sm complex is greater than the adsorption of IBU molecules by pure chitosan (Table 1). The adsorption of IBU molecules by the Sm-free chitosan is about 26.61%. Adsorption efficiency increases with increasing concentrations of Sm3+ ions contained in the chitosan. These results indicate that Sm3+ ions affect chitosan ability to adsorb IBU molecules. Chitosan–Sm–IBU with the Sm loading of 25 wt.% has adsorption efficiency of IBU molecules of 33.04%. This amount is almost the same with the adsorption efficiency of IBU (33.03%) by the chitosan–Sm–IBU with Sm3+ loading of 15 wt.%. This signifies that the Sm3+ loading in chitosan in the range of15–25 wt.% is optimum for the IBU adsorption onto the chitosan–Sm matrices. During adsorption of IBU molecules onto pure chitosan and chitosan– Sm, the carboxyl groups contained in the IBU form bonds with
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the hydroxyl groups from chitosan (Figs. 3B and 4). The addition of Sm3+ ions into chitosan will create bonding between the ions and the hydroxyl and amine groups of the chitosan (Fig. 3A). This will then increase the bond strength in these groups and thus they would become more reactive to bind the drug molecules. In addition, the modification of chitosan with the addition of Sm3+ ions will increase its reactivity, as noted by the increase in adsorption efficiency of IBU with the increasing content of Sm3+ ions in the chitosan. Determination of the IBU release profile of chitosan–Sm–IBU is necessary to understand the difference in profiles of pure chitosan with chitosan–Sm with various Sm loading. In addition, determination of the IBU release profile of the chitosan–Sm–IBU complex was performed to understand the influence of the presence of Sm3+ ions on the rate of IBU release from chitosan–Sm–IBU matrices. IBU release profiles from the chitosan–Sm–IBU matrices are shown in Fig. 7. The IBU adsorption pattern was conducted in triplicate. Standard deviation for each measurement in various Sm loading from 0 to 25 wt.% is 0.00043, 0.00099, 0.02607, 0.00073, 0.00171 and 0.00037, respectively. The in vitro release tests of IBU in the chitosan–Sm–IBU matrices were performed at pH 7.4, which is the condition in the intestinal fluid, for 24 h. In general, the drug release from chitosan occurs through a diffusion mechanism that swells the chitosan matrix [18]. In Fig. 7, it can be seen that the drug release profiles for pure chitosan and chitosan–Sm–IBU with five concentration variations of Sm3+ ions are quite similar. For all the drug release profiles, IBU is released slowly, reaching 50% in 8 hand perfectly separating in 24 h. Based on the graph in Fig. 7, it can be observed that the release mechanism of IBU from pure chitosan and chitosan–Sm–IBU depends on the diffusion mechanism. In this process, IBU molecules diffused slowly from pure chitosan and chitosan–Sm–IBU matrices. The drug release of IBU from both matrices begins with the swelling of chitosan–Sm as a drug coating material, followed by a slow diffusion of IBU molecules into the dissolution medium of the phosphate buffer pH 7.4 [13]. IBU diffusion is possible because of differences in the concentrations of IBU in the dissolution media of phosphate buffer pH 7.4 and the IBU in the chitosan–Sm. The slow rate of drug release of IBU could due to the strong hydrogen bonding and interaction between IBU molecules and the functional groups of the chitosan. Based on the graph, it can also be observed that the release of IBU from the chitosan–Sm–IBU matrices is slower than the release of IBU from pure chitosan. This could be due to the presence of Sm3+ ions that increase the reactivity of
Fig. 7. Drug release profile of ibuprofen in the chitosan–Sm–IBU complex, where 0 = chitosan, a = chitosan–Sm–IBU 2.5 wt.%, b = chitosan–Sm–IBU 5 wt.%, c = chitosan–Sm–IBU 10 wt.%, d = chitosan–Sm–IBU 15 wt.%, e = chitosan–Sm–IBU 25 wt.%. Standard deviation with confidence level of 95.0% for each measurement in various Sm loading from 0 to 25 wt.% are 0.00043, 0.00099, 0.02607, 0.00073, 0.00171 and 0.00037, respectively.
hydroxyl groups of chitosan, thus producing a strong hydrogen bond between hydroxyl groups from chitosan and the carboxyl group of IBU. For all variations, the drug release profile continues to rise to reach an equilibrium point after 12 h. This finding is consistent with the human digestive system time of 8 h. IBU released from the chitosan–Sm–IBU matrices in the phosphate buffer at pH 7.4 enters the matrices of pure chitosan and chitosan–Sm–IBU. These matrices expand and swell causing IBU molecules diffuse into the phosphate buffer until the concentration of IBU in the phosphate buffer is the same as the concentration of IBU in the pure chitosan and chitosan–Sm–IBU matrices at the time of synthesis. The addition of Sm3+ ions into chitosan was intended to allow the drug release from chitosan–Sm–IBU could be monitored through the changes in fluorescent intensity. The relationship between these intensity changes and the cumulative amount of IBU released from the chitosan–Sm–IBU matrices is shown in Fig. 8. Fluorescent intensity of the drug release samples was measured according to the characteristics of Sm3+ ions, which emit fluorescence with the 4G5/2 ? 6H7/2 transition at 590 nm and the excitation wavelength of 295 nm. Based on Fig. 8, it can be seen that the fluorescent intensity for all variations of the chitosan–Sm– IBU matrices increases with the cumulative amount of IBU released from the chitosan–Sm–IBU matrices. This finding is similar to the results reported by Yang et al. [13]. When IBU molecules are adsorbed onto the surface of chitosan, hydrogen bonding occurs between the hydroxyl group of chitosan and the carboxyl group of IBU causes the Sm3+ ions bonding with the hydroxyl groups in chitosan to weaken [13]. Therefore, fluorescent intensity of the Sm3+ ions in the matrices of chitosan–Sm–IBU will be weakened. In the process of drug release, IBU slowly detaches from the matrices indicated by weakening of the bond between chitosan hydroxyl group and IBU carboxyl group. At the same time, the bond between the Sm3+ions and hydroxyl groups in chitosan will rebound. This will cause the fluorescent intensity to increase with the cumulative amount of IBU released from the chitosan–Sm–IBU matrices. At the beginning, the cumulative release pattern of IBU (below 40%) from the chitosan–Sm are similar. Later on, the pattern changes after the cumulative release of IBU is greater than 45%. This may be due to the similar surface as well as interaction between IBU and chitosan and/or chitosan–Sm. The Sm-modified chitosan (2.5 wt.%) gives the highest intensity compared to the others chitosan–Sm (Fig. 8). The cumulative release of IBU (5%) from chitosan–Sm (2.5 wt.%) occurs at a fluorescence intensity of 4221 a.u., then slightly increases and reaches 4366 a.u. for the cumulative release of IBU (38.7%). Furthermore, increasing the cumulative release of IBU until 90.8% take place at a fluorescence
Fig. 8. Comparison of fluorescence intensity for drug release activity, where a = chitosan–Sm–IBU 2.5 wt.%, b = chitosan–Sm–IBU 5 wt.%, c = chitosan–Sm–IBU 10 wt.%, d = chitosan–Sm–IBU 15 wt.%, e = chitosan–Sm–IBU 25 wt.%.
E. Kusrini et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 120 (2014) 77–83
intensity of 6431 a.u. On the other hand, the cumulative release of IBU from chitosan–Sm 25 wt.% gradually increases and reaches 47.3% at a fluorescence intensity of 2952 a.u. After that, the cumulative release of IBU is faster and sharper compared to that of the others of matrices, even though it still occurs below of that the chitosan–Sm 2.5 wt.%. Finally, the cumulative release of IBU (100%) takes place at fluorescence intensities of 6653; 5701; 4857; 5261 and 6100 a.u., respectively for the chitosan–Sm (2.5– 25 wt.%) (see again Fig. 8). It is found that the lowest concentration of Sm loading in chitosan–Sm (2.5 wt.%) has the hypersensitive fluorescence intensity higher than the others, even though the adsorption efficiency of chitosan–Sm 2.5 wt.% is lower (29.75%) compared to the chitosan–Sm 25 wt.% (33.04%). Conclusion The chitosan–Sm complexes with different concentrations of Sm from 0 to 25 wt.% have been successfully synthesized by the impregnation method. The concentration variation of Sm loading in chitosan was introduced to understand the influence of Sm3+ ion content on chitosan ability to adsorb a drug and the drug release process. The addition of Sm3+ ions into chitosan enhances its ability to adsorb IBU molecules as a model drug. The results show that the hypersensitive fluorescence intensity of chitosan– Sm (2.5 wt.%) is higher than the others, even though its adsorption efficiency is lower (29.75%) compared to that of chitosan–Sm 25 wt.% (33.04%). Chitosan and Sm could be used to modify a drug release profile, drug adsorption, and drug distribution in the body so that medications will work optimally and can be monitored. Fluorescence properties of the Sm3+ ions could be used to indicate IBU release from the chitosan–Sm–IBU matrices; the change in fluorescent intensity takes place in the hypersensitive peak at 590 nm. Chitosan–Sm 25 wt.% demonstrated the highest adsorption efficiency of IBU (33.04%). Based on bioactivity of chitosan and fluorescent properties of Sm3+ ions, chitosan–Sm complexes would be promising for potential applications in the field of drug delivery and disease therapy.
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