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chitosan composite beads for diclofenac sodium delivery
International Journal of Biological Macromolecules 127 (2019) 594–605
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of pH-sensitive Fe3O4@C/carboxymethyl cellulose/chitosan composite beads for diclofenac sodium delivery Xiaoxiao Sun, Jiafeng Shen, Di Yu, Xiao-kun Ouyang ⁎ School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, PR China
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Article history: Received 13 December 2018 Received in revised form 31 December 2018 Accepted 28 January 2019 Available online 30 January 2019 Keywords: Fe3O4@C Carboxymethyl cellulose Chitosan pH-sensitive carrier
a b s t r a c t In this study, we prepared pH- and magnetism-responsive Fe3O4@C/carboxymethyl cellulose (CMC)/chitosan composite microbeads for controlled release of diclofenac sodium (DS) to (i) prevent complete drug release in gastric area, (ii) maintain blood drug concentration in a specific part of the body, (iii) reduce drug administration time and systemic drug toxicity, and (iv) improve drug efficacy. Through one-step solvent thermal treatment, a polyethylene glycol layer was wrapped into Fe3O4 nanoparticles. Then, Fe3O4@C nanoparticles were incorporated into CMC matrix and coated with chitosan layer via a self-assembly technique to form core–shell polyelectrolyte complexes (PECs). The composite beads were characterized by SEM, TEM, FT-IR spectrometry, and TGA. In addition, the effect of different concentrations of Fe3O4@C, CMC, aluminum chloride (AlCl3), and chitosan on the swelling process of composite beads, DS loading, and controlled release behavior was systematically studied. DS encapsulation efficiency in Fe3O4@C/CMC/chitosan beads reached 70.8 ± 0.65% at concentrations of 0.1% Fe3O4@C, 3% CMC, 3% AlCl3, and 1% chitosan. The beads showed a higher swelling index in phosphate buffer at pH 7.4 and 6.8 than at pH 1.2. The composite beads revealed excellent pH-sensitive in vitro drug release profiles and prevented burst release in the gastrointestinal tract. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The development of drug carriers, which directly impact the effects and delivery process of drugs, has received increased attention. Oral administration is currently the most common route of drug delivery. The drug can be delivered to the stomach, small intestine, and colonspecific sites in succession [1–3]. However, oral administration is susceptible to gastrointestinal pH, enzyme system, and liver first-pass effect, resulting in a in drug utilization and therapeutic effect [4]. Thus, for designing new oral drug delivery systems, it is essential to overcome these limitations besides controlling drug release and reducing the required dose [5–7]. Controlled drug release and targeted delivery carriers play important roles in maintaining blood drug concentration in a specific part of the body, thus reducing drug administration time and systemic drug toxicity, and improving drug efficacy [8,9]. The materials used as sustained release carriers of drugs include synthetic macromolecules [10–12] (e.g., polylactic acid, polycaprox, polyacrylate) and natural macromolecules [13–16] (e.g., gelatin, cellulose, chitosan). Synthetic macromolecular carriers are often not biocompatible and sometimes
⁎ Corresponding author at: School of Food and Pharmacy, Zhejiang Ocean University, Haida South Load 1#, Lincheng, Zhoushan 316022, PR China. E-mail address: [email protected] (X. Ouyang).
https://doi.org/10.1016/j.ijbiomac.2019.01.191 0141-8130/© 2019 Elsevier B.V. All rights reserved.
contain toxic impurities. Natural polymers have no such disadvantages [14,17]. Carboxymethyl cellulose (CMC) is a naturally anionic water-soluble polysaccharide [18,19]. It is widely used in the preparation of drug carriers owing to its ability to form spherical gel beads in the presence of Fe3+ or Al3+ [6]. Chitosan (CS) is one of the most abundant cationic marine biopolymers, and it exhibits a pH-sensitive property. It has been extensively used and studied for biomedical applications [20,21], such as wound dressing, tissue engineering, and drug delivery systems. In addition, chitosan can be degraded by enzymes, especially lysozyme, ensuring its biological safety [22]. However, owing to the poor mechanical strength and pH sensitivity of CMC and chitosan, drug controlled delivery systems based on CMC or chitosan are limited [23,24]. A possible solution to overcome the above disadvantages and develop a better gastrointestinal drug loading system based on CMC and chitosan is to form polyelectrolyte complexes (PECs) [25] between the carboxyl groups of CMC and the amino groups of chitosan [26,27]. Nevertheless, relying only on pure PEC layer cannot completely overcome the disadvantage of insufficient strength of materials. Fe3O4 magnetic nanoparticles have been widely used in biomedical research areas, such as drug delivery, cancer diagnosis and treatment, and microbial purification [28,29]. It is assumed that Fe3O4 magnetic nanoparticles, which have unique magnetic properties, low toxicity, and biocompatibility, can be incorporated into the hydrogel matrix to improve mechanical strength [30,31]. However, because of the dispersion of pure Fe3O4
X. Sun et al. / International Journal of Biological Macromolecules 127 (2019) 594–605 Table 1 Quantity of reactants for the preparation of Fe3O4@C/CMC/CS nanocomposite beads. Samples S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Fe3O4@C (wt%)
CMC (wt%)
CS (wt%)
AlCl3 (wt%)
0 0.025 0.050 0.100 0.100 0.100 0.100 0.100 0.100 0.100
3 3 3 3 2 4 3 3 3 3
1 1 1 1 1 1 0.50 1.50 1 1
3 3 3 3 3 3 3 3 2 4
magnetic nanoparticles [32–34], there is a possibility that these particles may not be removed easily after entering the gastrointestinal tract and may get deposited on the gastrointestinal surface. Therefore, Fe3O4 nanoparticles must be embellished to some extent for further use in drug delivery. This problem can be solved by modifying the structure of Fe3O4 nanoparticles with a thick carbon layer and abundant functional groups [35–37]. The high porosity and abundant –OH groups provide Fe3O4@C a superior adsorption performance on substances through electrostatic attractions. Notably, Fe3O4@C nanospheres could still be well dispersed in water and easily separated by an external magnet because of their excellent hydrophilic property and superparamagnetism [35,38]. Based on the above background, layer-by-layer (LBL) self-assembly technology [39] was adopted in this experiment to prepare dual-core shell pH-sensitive Fe3O4@C/CMC/CS microspheres. The stability and spheronization of the whole system were greatly improved by adding Fe3O4@C nanospheres to the CMC matrix. Through ionic interactions between carboxylate anions of CMC and cationic amine groups of chitosan, the pH-sensitive carrier for loading and delivery of diclofenac sodium (DS; an anti-inflammatory drug) can be expected to be used for gastrointestinal tract applications of controlled-release drug carrier. The developed pH-sensitive Fe3O4@C/CMC/CS microbeads were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrophotometry (FTIR), and thermogravimetric analysis (TGA), and the drug loading efficiency and loading content were estimated. The pH sensitivity of the developed Fe3O4@C/CMC/CS microbeads was examined from the point of
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measuring their swelling degree and in vitro drug release under simulated gastrointestinal tract conditions. In brief, the generated composite PECs could be expected to improve the encapsulation efficiency of DS, minimize its loss in stomach, and control its release in a localized colon region. 2. Materials and methods 2.1. Materials Carboxymethyl cellulose (MW = 90,000 (DS = 0.7), viscosity: 50–100 mPa.s), chitosan (deacetylation degree N95%, viscosity: 100–200 mPa.s), ferrocene (Fe(C5H5)2, purity of 99%), diclofenac sodium, aluminum chloride (AlCl3), polyethylene glycol (PEG, MW = 1000), hydrogen peroxide (H2O2, purity of 30%), acetone (C3H6O, purity of 99%), disodium hydrogen phosphate, sodium dihydrogen phosphate, and hydrochloric acid were purchased from Aladdin Chemical Co. Ltd. (Shanghai, China). All these reagents and chemicals were of analytical grade. 2.2. Preparation of Fe3O4@C According to the literature [38], Fe3O4@C was synthesized through one-step solvent thermal treatment, and 1:1 was adopted as the optimal proportion of composite. After accurately weighing 1.6 g ferrocene and 1.6 g PEG in a 100-mL volumetric flask, 70 mL of acetone was added and blended well until complete dissolution of ferrocene and PEG. After magnetic stirring (120 rpm) for 30 min, H2O2 (4 mL) was dropped slowly into the solution and continuously stirred for 2 h. Then, the entire solution was transferred from the flask to a 100-mL Teflon-lined stainless-steel autoclave and maintained at high temperature (200 °C) for 48 h. As the vessel cooled, the precipitates were collected via magnetic separation and washed at least three times with alcohol. Finally, the black products (Fe3O4@C) were dried at 60 °C in a vacuum oven for 12 h. 2.3. Preparation of Fe3O4@C/CMC/CS composite beads The DS delivery system was obtained by mixing the prepared CMC solution with Fe3O4@C and DS, followed by coating with chitosan. The formulation composition for preparation of Fe3O4@C/CMC/CS is given in Table 1. Typically, a weighted amount of CMC was dissolved in hot
Scheme 1. Preparation of pH-sensitive Fe3O4@C/CMC/CS composite beads.
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distilled water at 60 °C with stirring for 1 h until a homogenous and uniform solution was obtained. Then, a predetermined amount of Fe3O4@C was dispersed in distilled water and added slowly to the above CMC aqueous solution. The viscous liquid was prepared by ultrasonic dispersing technology for 1 h to obtain a uniform dispersion system. Subsequently, the suspension containing CMC and Fe3O4@C was extruded in the form of droplets using a 5-mL syringe (inside diameter of around 10 mm) into 100 mL of a certain concentration of AlCl3 solution at an invariable dropping rate at 25 °C. The formed spherical Fe3O4@C/CMC beads were
allowed to crosslink for 1 h under slow stirring to solidify the core, followed by rinsing with distilled water to remove unreacted AlCl3 on the surface of the beads. Finally, the semi-products were immersed in a certain concentration of chitosan solution for 2 h, resulting in instantaneous formation of core-shell beads due to electrostatic interactions between chitosan and CMC. These coated beads were washed with distilled water to remove excess of unreacted chitosan and dried in a vacuum oven at 40 °C for further use. The presumptive mechanistic route for the formulation of Fe3O4@C/CMC/CS beads is shown in Scheme 1.
Fig. 1. Scanning electron microscopy (SEM) images of Fe3O4@C (a), Fe3O4@C/CMC/CS beads (b), Fe3O4@C/CMC (c) and its larger image (d), Fe3O4@C/CMC/CS powder (e) and its larger image (f).
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Fig. 2. Transmission electron microscopy (TEM) images of Fe3O4@C (a), Fe3O4@C/CMC (b), Fe3O4@C/CMC/CS powder (c), and Fe3O4@C/CMC/CS beads (d).
The preparation of drug-loading Fe3O4@C/CMC/CS beads was the same as above. A certain concentration (1 mg/mL) of DS solution was transferred into the mixture in the same way to obtain a final volume of 100 mL.
predetermined time intervals, the excess surface-adhered liquid drops were removed by blotting with a filter paper, and the swollen beads were weighed again immediately. The swelling ratios of the microcapsules were calculated using Eq. (1):
2.4. Characterization Swelling ratio ð%Þ ¼ The surface morphology of the microgel beads was studied with a SEM (S-4800, Hitachi Limited Ltd., Japan). For SEM, all samples were coated with a thin gold film under vacuum prior to microscopy analysis, and these samples were viewed using an accelerating voltage of 15 kV at the appropriate magnification. TEM images of Fe3O4@C powder, Fe3O4@C/CMC, and Fe3O4@ C/CMC/CS beads were obtained using Lorentz Transmission Electron Microscope (TEM, JEM-2100, JOEL, Japan). FT-IR (Tensor II, Bruker, Germany) was used to characterize the functional groups of the materials. In each case, the powder samples were placed into KBr (spectrum pure) pellet, and then the spectra were scanned and recorded from 400 to 4000 cm−1 at room temperature (25 °C). Furthermore, TGA of all samples was performed by employing a TGA (Pyris Diamond TG/DTA, Perkin-Elmer, USA) at a heating rate of 20 °C/min from 20 to 800 °C under N2 atmosphere.
W t −W 0 100% W0
ð1Þ
where Wt is weight of the swollen microcapsules at time t and W0 is the initial weight of microcapsules.
2.5. Swelling ratio studies The swelling behavior was studied by measuring the percentage of water uptake by microcapsule beads via the immersing method. Accurately weighed 0.1 g of dried beads were allowed to swell in 50 mL of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated colonic fluid (SCF, pH 7.4) at 37 °C. At
Fig. 3. FT-IR spectra of (a) CMC, (b) chitosan, (c) Fe3O4@C, (d) Fe3O4@C/CMC, and (e) Fe3O4@C/CMC/CS.
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where We is the amount of drug in the weighted amount of microcapsules in equilibrium and W0 is the amount of drug that was first added during preparation procedure. 2.7. In vitro drug release studies For in vitro DS release studies from the loaded microcapsules, three different pH solutions (SGF, SIF, and SCF) were investigated. In each experiment, 0.1 g of dried microcapsule beads were immersed in SGF (2 h) and then transferred to SIF (4 h) or SCF (4 h) at 37 °C in a water bath at 100 rpm to ensure that all the encapsulated DS was extracted by the used pH buffer. At predetermined time intervals, approximately 3 mL of the buffer solution was removed and filtered for assaying at 284 nm using an UV spectrophotometer. The removed solution was replaced with the same volume of fresh PBS. The amount of drug released from the beads was determined by using a calibration plot curve obtained under the same conditions. Fig. 4. TGA of CMC (a), Fe3O4@C (b), Fe3O4@C/CMC (c), and Fe3O4@C/CMC/CS beads (d).
3. Results and discussion
2.6. Drug loading studies
3.1. Analysis of shape and appearance
The loading of DS into all the microcapsule beads was achieved by the wrapped method as mentioned above (preparation of microcapsules). Then, the dried drug-loaded beads were crushed in a mortar with a pestle, followed by sifting of the polymeric powder (0.1 g) to an Erlenmeyer flask using a 200-mesh sieve. Subsequently, 50 mL (pH 7.4) of phosphate-buffered solution (PBS) was added with shaking at 25 °C for 24 h in a water bath to ensure complete extraction of DS from the beads. After 1 h, precipitated gels were filtered; DS was analyzed by using a UV spectrophotometer at a wavelength of 284 nm using a calibration curve and PBS (pH 7.4) as the blank. The percentage of entrapment efficiency was then calculated using Eq. (2):
To analyze Fe3O4@C/CMC/CS beads and determine the shape and appearance of the constituent components further, Fe3O4@C, Fe3O4@ C/CMC, and Fe3O4@C/CMC/CS powders were examined respectively by SEM and TEM, as shown in Figs. 1 and 2. As shown in Scheme 1 and Fig. 1, magnetic iron oxide particle (Fig. 1a), which was surrounded with PEG, showed more centralized magnetism. The wet beads (Fe3O4@C/CMC/CS) were almost spherical and smooth, with an average particle size of approximately 3 mm and gray color (that changed slowly to black with increasing amount of Fe3O4@C). The particle size of composite microspheres when dried at 40 °C tended to become smaller to approximately 1.3 mm. The surface of microspheres became rough and porous (Fig. 1b). This could be because the process of drying led to partial collapse of the polymeric gel network structure.
Drug encapsulation efficiencyð%Þ ¼
We 100% W0
ð2Þ
Fig. 5. Effect of Fe3O4@C concentration (a), CMC concentration (b), AlCl3 concentration (c), and chitosan concentration (d) on DS encapsulation efficiency of Fe3O4@C/CMC/CS beads.
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Fig. 6. Effects of Fe3O4@C concentrations on the swelling ratio of Fe3O4@C/CMC/CS beads in SGF (a), SIF (b), and SCF (c). Effects of CMC concentrations on the swelling ratio of Fe3O4@ C/CMC/CS beads in SGF (d), SIF (e), and SCF (f).
Comparison of the SEM images of Fe3O4@C/CMC (Fig. 1c) and Fe3O4@C/CMC/CS (Fig. 1e) showed that the surface of Fe3O4@ C/CMC/CS beads was less wrinkled. This was attributed to the assembly of chitosan on the Fe3O4@C/CMC layer to form a neat, tight, and flat surface by interactions between CMC and chitosan. In addition, the measurement of diameter of Fe3O4@C/CMC/CS wet bead constricts during the chitosan coating process was an acknowledgement of the interaction. It can be seen from Fig. 1d that Fe3O4@C particles were wrapped into the CMC colloid. A magnified version of Fe3O4@C/CMC/CS (Fig. 1f) after the assembly of chitosan on the Fe3O4@C/CMC layer showed that the surface of composite microspheres had numerous wrinkled areas, which favored water and tissue fluid infiltration. TEM was used to determine the crystallographic and morphological discrepancy of these as-prepared beads with high spatial resolution (Fig. 2a–d). The Fe3O4@C inner cores (Fig. 2a) were magnetic black
globules that were sealed uniformly with PEG. When the inner cores were wrapped into CMC to solidify in AlCl3 solution, the magnetic black globule attracted each other to form a more compact and stable sphere (Fig. 2b). As shown in Fig. 2c, after coating with chitosan, the interfacial interactions caused shrinking of the layers to form a more stable system. 3.2. FT-IR FT-IR analysis of raw CMC, chitosan, Fe3O4@C, Fe3O4@C/CMC, and Fe3O4@C/CMC/CS beads was carried out, and the data are shown in Fig. 3. FT-IR analysis was performed to verify that CMC and chitosan were coated on the system during material preparation. In the spectrogram of raw CMC (Fig. 3a) and chitosan (Fig. 3b), the peaks at 3445 cm−1 and 3430 cm−1 were respectively caused by the stretching
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Fig. 7. Effects of AlCl3 concentrations on the swelling ratio of Fe3O4@C/CMC/CS beads in SGF (a), SIF (b), and SCF (c). Effects of chitosan concentrations on the swelling ratio of Fe3O4@ C/CMC/CS beads in SGF (d), SIF (e), and SCF (f).
vibration of O\\H in CMC, and O\\H and N\\H in chitosan. A broad asymmetrical band was observed at 1645 cm−1 and a narrower symmetrical band at 1420 cm−1, which corresponded to the basic characteristic functional group –COO−. In the chitosan spectrum, the absorption bands at 1640 cm−1 corresponded to carbonyl stretching of the secondary amide. The bands between 1300 and 1460 cm−1, and at 1000–1200 cm−1 are assigned to C\\N and C\\O stretching on the polysaccharide skeleton, respectively [40]. Obvious changes were observed in the FT-IR spectrum of Fe3O4@ C/CMC/CS core–shell beads compared with those of the other samples. In the Fe3O4@C/CMC/CS microspheres, movement to a lower frequency of 3425 cm−1 indicated the formation of hydrogen bonds between the carboxyl group of CMC and the hydroxyl group of chitosan, as well as the superposition of N\\H and O\\H groups. The line (c) demonstrates a peak at 596 cm−1, which is attributed to Fe\\O
bond [41]. The bands at 1638 cm−1 were attributed to –C_O bond. These bands combined with the presence of the stretching vibration of O\\H illustrated that the carbon layer and abundant functional groups were wrapped into Fe3O4 magnetic nanoparticles [42]. As can be seen in Fig. 3d–e, a new absorption band was observed at 1480 cm−1, which can be interpreted in successfully coated chitosan layer. Some small peaks increased in strength between 1400 cm−1 and 1600 cm−1, which indicated the form of PEC structure. 3.3. TGA Thermogravimetric curves determined for CMC, Fe3O4@C, Fe3O4@ C/CMC, and Fe 3 O4 @C/CMC/CS microcapsule beads are shown in Fig. 4. From the degradative curves of Fe3O4@C (Fig. 4b), it was evident that the magnetic iron oxide was successfully wrapped into
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PEG. Since –OH and –CH of Fe3O4@C were carbonized, the weight of the sample was reduced slightly when the temperature increased. Similarly, the weight curves of Fe3O4@C/CMC (Fig. 4c) and Fe3O4@ C/CMC/CS (Fig. 4d) showed a gradual decline to 80%. This is due to the degradation of the polymer and hydrogen-bound water in the temperature range of 80–200 °C, which forms the polysaccharide structure of CMC and CS. In addition, a uniform and smooth decline in weight loss at 250–400 °C in Fe 3O 4 @C/CMC and Fe3 O 4@ C/CMC/CS was observed. This may be partly attributed to the degradation and decomposition of organic skeletal structure, amino groups, and other functional groups. By comparing the curves of Fe3O4@C/CMC and CMC (Fig. 4a), it was observed that Fe3O4@C particles are wrapped into CMC and can enhance the thermal stability of the whole system. At a temperature of 500 °C or higher, the remaining material was carbonized completely. Comparison of the degradation processes of Fe3O4@C/CMC and Fe3O4@C/CMC/CS showed that the weight loss rate for the latter was much higher than that for the former at the same condition. This indicated that chitosan coated successfully and penetrated deeply into the surface of Fe3O4@C/CMC by forming a PEC through polar interactions between carboxylate groups (CMC) and amino groups (chitosan). In addition, Fe3 O 4 @ C/CMC/CS composite beads were more thermally stable than Fe 3 O4 @C/CMC. Given the above, it can be suggested that the Fe3O4@C/CMC/CS drug delivery system would be thermally stable at the physiological temperature of the human body. 3.4. DS encapsulation efficiency Drug loading efficiency is a crucial parameter to determine the effectiveness of drug delivery vehicle [43,44]. Excellent drug loading rates may effectively increase the application of drug-carrying systems and reduce the waste of bulk drug substances. In the present study, we
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investigated the encapsulation efficiency of DS in diverse Fe3O4@ C/CMC/CS beads by comparing the effects of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan. In the experiment, the initial concentration of DS was constant and equivalent (1 g/L). The percentage of DS encapsulation was around 55–80% and it varied with the concentration of raw materials as shown in Fig. 5. The encapsulation of hydrophilic molecules such as DS remains a significant challenge, because these drugs exhibit a strong tendency to leak to the external aqueous phase. The factors that affect the encapsulation of hydrophilic molecules include concentrations of matrix materials and crosslinker during encapsulation process, indicating that Fe3O4@C/CMC/CS system can be an excellent drug carrier. DS was successfully loaded into Fe3O4@C/CMC/CS beads and the encapsulation efficiency (%) increased from 52.2 ± 0.79% to 70.8 ± 0.65% with increase in concentration of Fe3O4@C from 0.025% to 0.1% (CMC: 3%; AlCl3: 3%; CS: 1%), as shown in Fig. 5a. This illustrates the importance of Fe3O4@C particles in the whole system, because they showed concentrated magnetism and formed a steady inner core structure. However, if the concentration of Fe3O4@C (i.e. 0.2%) is increased continuously, the microsphere structure will show a tendency to burst and rupture. This could be because of the overweight of Fe3O4@C particles and magnetic interaction. The compound beads were hard to solidify when the concentration of CMC was low (i.e. 1–2%). The colloid with excessive levels (exceeds 5%) of CMC was too thick to form droplets; therefore, concentrations of CMC from 3%–5% were adopted to analyze the effect of CMC concentration. The results (Fig. 5b) showed that the drug loading efficiency of 3% CMC reached a peak (70.8 ± 0.65%), and by increasing the concentration of CMC to 4% and 5%, the encapsulation efficiency (%) reached 69.53 ± 1.10% and 65.93 ± 0.70%, respectively. A possible explanation for this profile is that the thick matrix has a more compact structure and is easy to crosslink in AlCl3 solution. However, when the concentration of CMC was increased, the dissolution and swelling of CMC had an effect on the solubility of DS. Thus, the encapsulation
Fig. 8. Effects of Fe3O4@C concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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efficacy (%) showed a slightly downward trend and the gel turned turbid in the preparation process. Taken together, the optimum concentration of CMC as the gel stroma was 3%, considering the encapsulation efficacy (%), homogeneity of dosage, and economic factors. Regarding the effect of cross-linking agent (AlCl3) concentration, the rate of interaction of CMC beads and AlCl3 solution increased with increase in concentration of cross-linking agent, leading to a lesser chance of the drug oozing out. When the AlCl3 concentration increased from 2% to 4%, the encapsulation efficacy (%) increased from 62.5 ± 0.52 to 75.1 ± 0.82, as shown in Fig. 5c. Nevertheless, the encapsulation efficacy (%) did not continue to increase, despite the increase in AlCl3 concentration. This could be attributed to the fact that the overall surface area of CMC beads remained unchanged. Finally, when increasing the CS concentration from 0.5% to 1.5% (Fig. 5d), the loading efficiency increased from 62.6 ± 0.94% to 77.5 ± 0.76%. This indicates that the NH3+ group of chitosan exhibited electrostatic interactions with the –COO– group of CMC during PEC formation process, and the thicker chitosan layer (1.5%) resulted in entrapment of drugs in the more thicker PEC shell, which was better than paper shell beads. It is also reasonable to assume that the higher the CS concentration, the thicker the crust of beads and lower the drug loss during the preparation process (long contact and more washing time during the coating process). Similarly, the limited surface groups led to restricted encapsulation efficacy (%). In summary, the concentration of Fe3O4@C, CMC, AlCl3, and chitosan has an effect on the association of DS to Fe3O4@C/CMC/CS beads. The encapsulation efficiency of DS in Fe3O4@C/CMC/CS beads was more balanced (70.8%) at 0.1% Fe3O4@C, 3% CMC, 3% AlCl3, and 1% chitosan. 3.5. Swelling studies Swelling has been considered one of the most important properties of bio-nanocomposite hydrogel beads that determine the release rate of
the encapsulated drug [45,46]. It indicates the easiness and speed of a liquid to penetrate the beads and affect drug diffusion. The swelling degree is used to determine the ability of hydrogels to soak up water, which can be quantitatively analyzed by increasing the size and weight of beads. To investigate this further, we must consider the effects of factors such as solubility of polymers, porosity, nature of ionic groups, and formation of ionic bond. As shown in Figs. 6 and 7, the effects of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan on the swelling behavior of Fe3O4@C/CMC/CS beads were determined at pH of 1.2, 6.8, and 7.4. The swelling behavior was dependent on the following factors: (i) pH, (ii) time, and (iii) concentrations of raw materials. Swelling increased and the growth rate decreased with time, and then gradually reached the maxima. Some beads began to disintegrate. All investigated Fe3O4@C/CMC/CS beads with different contents maintained their structures in acidic environment (SGF). However, with increase in pH, the swelling rate increased by dozens of times (1000–3000%) and their structures were destroyed. This pH-dependent swelling behavior can be explained by the maintenance of CMC–Al3+ complexes in microcapsule core. In addition, only a few chitosan amine groups were protonated in the exterior shell in SGF. The increased pH induced deprotonation of ionizable carboxylic groups on the CMC chain. Moreover, phosphate ions displaced Al ions within the beads after the shell was destroyed, leading to higher electrostatic repulsion between these charged groups. This subsequently causes an increase in osmotic pressure and amount of water penetrating into the molecules. Our study discusses the effect of different concentrations of Fe3O4@C particles, and the results are shown in Fig. 6a–c. Because of the frangibility and instability of pure CMC/chitosan beads, the experiment was conducted without regard to pure beads. At lower concentrations of Fe3O4@ C particles, the moisture-absorbing rate of Fe3O4@C/CMC/CS beads was faster, swelling ratio was lower, and the whole system collapsed rapidly due to inadequate material strength. With the increase in Fe3O4@C
Fig. 9. Effects of CMC concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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particles, Fe3O4@C/CMC/CS beads adsorbed more water and maintained the form with no changes in swelling state. A possible explanation for this is that Fe3O4@C particles could support the hydrogel network and enhance the mechanical strength of CMC matrix, which are important in drug loading. Notably, exorbitant concentrations of Fe3O4@C particles can result in overweight load and excessive magnetism, which in turn can lead to collapse of the whole system. This underscored the significance and indispensability of appropriate Fe3O4@C particles. Because low CMC concentrations (i.e. 1%, 2%) were too sparse to maintain the structure, we selected 3%–5% CMC concentrations to study the effect. Based on the data presented in Fig. 6d–f, CMC concentrations of 4% and 5% resulted in the highest swelling rates at pH 6.8 and 7.4, indicating that the concentration of CMC as the matrix played an important role in swelling performance. This is due to the reaction balance between –COO−, Al ions, and –NH2 at lower CMC concentrations, and the saturation of chelation between Al3+ and –COO– led to the formation of a dense nucleus. As the CMC concentration increased, an external layer was formed by PECs, wherein the amine groups of chitosan electrostatically interacted with a much larger number of carboxyl groups of CMC. As shown in Fig. 6d, under acidic conditions, 3% CMC beads were more easily swollen and maintained their structure and morphology in 6–8 h. The more the CMC chain was exposed, the more the amount of infiltrated water and the easier the disintegration of the swelling core. Based on these results, we speculated that increased CMC concentration leads to stronger water absorption capacity and a tighter and engorged inner core when other conditions are immobilized. As shown in Fig. 7a–c, under acidic conditions, the swelling ratio reached the peak rapidly (1450%) at lower concentration of AlCl3 (2%). In contrast, with increase in AlCl3 concentration, the swelling ratio tended to reduce. This could be because of rapid saturation of chelation
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between Al3+ and –COO−, subsequently resulting in the formation of a tough nucleus making it hard for water to penetrate. At 4% AlCl3 concentration, the site of bead surface had reached saturation, and predictably, higher AlCl3 concentration (N4%) only caused a slight increase. When the pH reached 6.8, a major increase in swelling ratio was observed. The penetrative process led to a slow swelling trend with increase of AlCl3 concentration, and the inner core structure became more stable. Similar trends were observed when the pH reached 7.4. However, the swelling ratio changed according to chitosan content (Fig. 7d–f). The inner core (CMC-Al3+ interface) tended to shrink at lower pH condition; thus, the outermost chitosan layer plays a major role in preventing water infiltration during the swelling process. The swelling capacity improved with increase in chitosan concentration from 0.5% to 1.5% at pH 6.8 and 7.4. This might be due to increased protonation of amine groups with increase in amount of –NH2 and decreased protonation with increase of pH. As the CS concentration increased, the swelling degree also increased, suggesting that the interaction between CMC and chitosan lines was reinforced. The intensive interaction is easy to cause an increase in osmotic pressure. Therefore, the swelling ratio of 1.0–1.5% chitosan ultimately attained a higher level at pH 6.8 and 7.4. Based on these results, the prepared Fe3O4@C/CMC/CS composite hydrogel beads can be applied for sustained drug delivery under different pH conditions. 3.6. Release studies A favorable drug control-release system is expected to retain enough amount of drug in the stomach and to release more amount of the drug in the intestine [47]. Therefore, orally administered drug-loaded polymeric formulations must have excellent pH sensitivity. However, a
Fig. 10. Effects of AlCl3 concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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sudden release of drug in the initial phase is not expected. To study the effects of Fe3O4@C, CMC, AlCl3, and chitosan concentrations on the release of DS from Fe3O4@C/CMC/CS composite beads, accurately weighed quantity (0.1 g) of dry composite beads were immersed in SGF (2 h) and transferred to SIF (4 h) or SCF (4 h) at 37 °C, and their cumulative release ratio was analyzed. The cumulative release ratio always maintained an extremely low value under acidic conditions (Fig. 8c). Within the error range allowed, there was a slight fluctuation in the release ratio of three different Fe3O4@C concentrations. After switching to the neutral (Fig. 8b) and alkaline (Fig. 8c) systems, the cumulative release ratio increased markedly, and higher concentration of Fe3O4@C corresponded to lower release. A possible explanation for this might be that the structure of the beads remained stable at higher Fe3O4@C concentrations leading to slower process of release and disintegration, mainly dependent on the magnetic properties. The cumulative release data shown in Fig. 9 indicated that as the concentration of CMC increased from 3% to 5%, a considerable decrease in DS release was observed at pH 1.2, 6.8, and 7.4. This result was attributed to the shrinking of CMC gel. More number of carboxyl groups of CMC are in the unionized form for sustained drug release in the stomach. To a certain extent, the higher the CMC concentration, the more compact the inner core, resulting in harder disintegration and decreased release ratio in phosphate buffer at pH 6.8 and 7.4. As shown in Fig. 10, when the AlCl3 concentration increased from 2% to 4%, the total amount of the released DS decreased in SGF, SIF, and SCF. The result showed that the drug release was higher at lower AlCl 3 concentration, because the more the number of Al3+ ions around CMC, the faster the formation of a tight inner core. The rigorous surface was caused by the complexation between Al 3+ and the hydroxyl and carboxyl groups of CMC, resulting in blockade of drug release. This finding is consistent with the previously
described swelling profile of Fe3O4@C/CMC/CS. In addition, we predicted that with the increase in AlCl3 concentration, a similar continuous decline in release ratio may not be observed because of the limited surface area. Finally, we also analyzed the effect of chitosan concentrations on drug release (Fig. 11). In acidic conditions, the degree of swelling and the protonation of amino groups of chitosan increased, resulting in easy release of drug from beads. The noticeable changes observed in SIF and SCF can be explained by the ionic interactions between the carboxyl groups of CMC and ammonium groups of chitosan by forming PEC, whose density increases with increasing concentrations of the polymers. In addition, the electrostatic interaction of drug-containing systems is easily broken at pH 6.8 and 7.4 than at pH 1.2. However, a shrinking tendency of the outer layer of chitosan was observed at pH 6.8 and 7.4, which ensured that the whole system remained in steady state for a period of time after increased water absorption and swelling of internal structure. Overall, the amount of DS released from Fe3O4@C/CMC/CS beads within 2 h was low (b10%) at pH 1.2. When the pH was changed from near neutral to alkaline, the eventual release rate showed a significant increase. The extent of protonation, properties of the model drug, swelling ratio, and drug release are all interlinked. A possible explanation might be that the microspheres began to swell after entering the liquid environment and external amino groups of chitosan are gradually ionized. Internal Fe3O4@C and CMC layer make the inner core tighter for preventing the entry of water molecules into the nucleus and for allowing the drug to leach out of the wet beads for longer periods. In the near neutral and alkaline phosphate buffer, the networks became more hydrophilic because of the formation of neutralized carboxyl in the CMC layer, leading to a collapsed state. Particularly, the release ratio was still maintained at 40–50% in the initial neutral and alkaline stages. These results reveal that the whole Fe3O4@C/CMC/CS composite
Fig. 11. Effects of chitosan concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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system can be an excellent controlled-release drug carrier in the pHsensitive environment of the gastrointestinal tract. 4. Conclusion Fe3O4@C particles serve as promising supporting materials for pHsensitive composite beads of Fe3O4@C/CMC/CS. The synthesized composite beads were characterized by SEM, TEM, FT-IR, and TGA. In addition, the drug encapsulation efficiency, pH-responsive swelling, and drug release behavior of the Fe3O4@C/CMC/CS system were studied based on the composition of the formulations, including the effect of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan. The swelling and release depended on the pH, time of incubation, and concentrations of raw materials. The beads showed a higher swelling ratio and release in phosphate buffer at pH 6.8 and 7.4 than at pH 1.2, and prevented a sudden release. These results indicated that Fe3O4@ C/CMC/CS composite beads in proper proportion can be an excellent and directed controlled-release drug carrier. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21476212) and Bureau of Science and Technology of Zhoushan (2017C41005). References [1] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (1) (2001) 1–20. [2] R. Masteiková, Z. Chalupová, Z. Sklubalová, Stimuli-sensitive hydrogels in controlled and sustained drug delivery, Medicina 39 (Suppl. 2) (2003) 19. [3] C. Isha, S. Nimrata, A.C. Rana, G. Surbhi, Oral sustained release drug delivery system: an overview, Int. Res. J. Pharm. 3 (5) (2012). [4] K.Y. Lee, S.H. Yuk, Polymeric protein delivery systems, Prog. Polym. Sci. 32 (7) (2007) 669–697. [5] G. Meera, A.T. Emilia, Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan–a review, J. Control. Release Off. J. Control. Release Soc. 114 (1) (2006) 1–14. [6] A.P. Rokhade, S.A. Agnihotri, S.A. Patil, N.N. Mallikarjuna, P.V. Kulkarni, T.M. Aminabhavi, Semi-interpenetrating polymer network microspheres of gelatin and sodium carboxymethyl cellulose for controlled release of ketorolac tromethamine ☆, Carbohydr. Polym. 65 (3) (2006) 243–252. [7] T. Coviello, P. Matricardi, C. Marianecci, F. Alhaique, Polysaccharide hydrogels for modified release formulations, J. Control. Release 119 (1) (2007) 5–24. [8] P. Bhusal, J. Harrison, M. Sharma, D.S. Jones, A.G. Hill, D. Svirskis, Controlled release drug delivery systems to improve post-operative pharmacotherapy, Drug Deliv. Transl. Res. 6 (5) (2016) 1–11. [9] J. Zhang, Q. Wang, A. Wang, In situ generation of sodium alginate/hydroxyapatite nanocomposite beads as drug-controlled release matrices, Acta Biomater. 6 (2) (2010) 445–454. [10] M. Kissel, P. Peschke, V. Subr, K. Ulbrich, J. Schuhmacher, J. Debus, E. Friedrich, Synthetic macromolecular drug carriers: biodistribution of poly[(N-2-hydroxypropyl) methacrylamide] copolymers and their accumulation in solid rat tumors, PDA J. Pharm. Sci. Technol. 55 (3) (2001) 191–201. [11] M. Kissel, P. Peschke, V. Šubr, K. Ulbrich, A.M. Strunz, R. Kühnlein, J. Debus, E. Friedrich, Detection and cellular localisation of the synthetic soluble macromolecular drug carrier pHPMA, Eur. J. Nucl. Med. Mol. Imaging 29 (8) (2002) 1055–1062. [12] X. Li, M.A. Kanjwal, L. Lin, I.S. Chronakis, Electrospun polyvinyl-alcohol nanofibers as oral fast-dissolving delivery system of caffeine and riboflavin, Colloids Surf. B: Biointerfaces 103 (2) (2013) 182–188. [13] A. Alves, S.G. Caridade, J.F. Mano, A.S. Rui, L.R. Rui, Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae, Carbohydr. Res. 345 (15) (2010) 2194–2200. [14] S. Jana, A. Gandhi, K.K. Sen, S.K. Basu, Natural polymers and their application in drug delivery and biomedical field, J. PharmaSciTech 1 (1) (2011) 16–27. [15] A.J. Smit, Medicinal and pharmaceutical uses of seaweed natural products: a review, J. Appl. Phycol. 16 (4) (2004) 245–262. [16] D.A. Giovanna Gomez, M. Mario, L. Paola, Marine derived polysaccharides for biomedical applications: chemical modification approaches, Molecules 13 (9) (2008) 2069–2106. [17] A. Anabela, S.G. Caridade, J.O.F. Mano, A. Rui Sousa, L. Rui Reis, Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae, Carbohydr. Res. 345 (15) (2010) 2194–2200. [18] M.S.M. Eldin, H.M. El-Sherif, E.A. Soliman, A.A. Elzatahry, A.M. Omer, Polyacrylamide-grafted carboxymethyl cellulose: smart pH-sensitive hydrogel for protein concentration, J. Appl. Polym. Sci. 122 (1) (2011) 469–479.
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International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac
Preparation of pH-sensitive Fe3O4@C/carboxymethyl cellulose/chitosan composite beads for diclofenac sodium delivery Xiaoxiao Sun, Jiafeng Shen, Di Yu, Xiao-kun Ouyang ⁎ School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, PR China
a r t i c l e
i n f o
Article history: Received 13 December 2018 Received in revised form 31 December 2018 Accepted 28 January 2019 Available online 30 January 2019 Keywords: Fe3O4@C Carboxymethyl cellulose Chitosan pH-sensitive carrier
a b s t r a c t In this study, we prepared pH- and magnetism-responsive Fe3O4@C/carboxymethyl cellulose (CMC)/chitosan composite microbeads for controlled release of diclofenac sodium (DS) to (i) prevent complete drug release in gastric area, (ii) maintain blood drug concentration in a specific part of the body, (iii) reduce drug administration time and systemic drug toxicity, and (iv) improve drug efficacy. Through one-step solvent thermal treatment, a polyethylene glycol layer was wrapped into Fe3O4 nanoparticles. Then, Fe3O4@C nanoparticles were incorporated into CMC matrix and coated with chitosan layer via a self-assembly technique to form core–shell polyelectrolyte complexes (PECs). The composite beads were characterized by SEM, TEM, FT-IR spectrometry, and TGA. In addition, the effect of different concentrations of Fe3O4@C, CMC, aluminum chloride (AlCl3), and chitosan on the swelling process of composite beads, DS loading, and controlled release behavior was systematically studied. DS encapsulation efficiency in Fe3O4@C/CMC/chitosan beads reached 70.8 ± 0.65% at concentrations of 0.1% Fe3O4@C, 3% CMC, 3% AlCl3, and 1% chitosan. The beads showed a higher swelling index in phosphate buffer at pH 7.4 and 6.8 than at pH 1.2. The composite beads revealed excellent pH-sensitive in vitro drug release profiles and prevented burst release in the gastrointestinal tract. © 2019 Elsevier B.V. All rights reserved.
1. Introduction The development of drug carriers, which directly impact the effects and delivery process of drugs, has received increased attention. Oral administration is currently the most common route of drug delivery. The drug can be delivered to the stomach, small intestine, and colonspecific sites in succession [1–3]. However, oral administration is susceptible to gastrointestinal pH, enzyme system, and liver first-pass effect, resulting in a in drug utilization and therapeutic effect [4]. Thus, for designing new oral drug delivery systems, it is essential to overcome these limitations besides controlling drug release and reducing the required dose [5–7]. Controlled drug release and targeted delivery carriers play important roles in maintaining blood drug concentration in a specific part of the body, thus reducing drug administration time and systemic drug toxicity, and improving drug efficacy [8,9]. The materials used as sustained release carriers of drugs include synthetic macromolecules [10–12] (e.g., polylactic acid, polycaprox, polyacrylate) and natural macromolecules [13–16] (e.g., gelatin, cellulose, chitosan). Synthetic macromolecular carriers are often not biocompatible and sometimes
⁎ Corresponding author at: School of Food and Pharmacy, Zhejiang Ocean University, Haida South Load 1#, Lincheng, Zhoushan 316022, PR China. E-mail address: [email protected] (X. Ouyang).
https://doi.org/10.1016/j.ijbiomac.2019.01.191 0141-8130/© 2019 Elsevier B.V. All rights reserved.
contain toxic impurities. Natural polymers have no such disadvantages [14,17]. Carboxymethyl cellulose (CMC) is a naturally anionic water-soluble polysaccharide [18,19]. It is widely used in the preparation of drug carriers owing to its ability to form spherical gel beads in the presence of Fe3+ or Al3+ [6]. Chitosan (CS) is one of the most abundant cationic marine biopolymers, and it exhibits a pH-sensitive property. It has been extensively used and studied for biomedical applications [20,21], such as wound dressing, tissue engineering, and drug delivery systems. In addition, chitosan can be degraded by enzymes, especially lysozyme, ensuring its biological safety [22]. However, owing to the poor mechanical strength and pH sensitivity of CMC and chitosan, drug controlled delivery systems based on CMC or chitosan are limited [23,24]. A possible solution to overcome the above disadvantages and develop a better gastrointestinal drug loading system based on CMC and chitosan is to form polyelectrolyte complexes (PECs) [25] between the carboxyl groups of CMC and the amino groups of chitosan [26,27]. Nevertheless, relying only on pure PEC layer cannot completely overcome the disadvantage of insufficient strength of materials. Fe3O4 magnetic nanoparticles have been widely used in biomedical research areas, such as drug delivery, cancer diagnosis and treatment, and microbial purification [28,29]. It is assumed that Fe3O4 magnetic nanoparticles, which have unique magnetic properties, low toxicity, and biocompatibility, can be incorporated into the hydrogel matrix to improve mechanical strength [30,31]. However, because of the dispersion of pure Fe3O4
X. Sun et al. / International Journal of Biological Macromolecules 127 (2019) 594–605 Table 1 Quantity of reactants for the preparation of Fe3O4@C/CMC/CS nanocomposite beads. Samples S1 S2 S3 S4 S5 S6 S7 S8 S9 S10
Fe3O4@C (wt%)
CMC (wt%)
CS (wt%)
AlCl3 (wt%)
0 0.025 0.050 0.100 0.100 0.100 0.100 0.100 0.100 0.100
3 3 3 3 2 4 3 3 3 3
1 1 1 1 1 1 0.50 1.50 1 1
3 3 3 3 3 3 3 3 2 4
magnetic nanoparticles [32–34], there is a possibility that these particles may not be removed easily after entering the gastrointestinal tract and may get deposited on the gastrointestinal surface. Therefore, Fe3O4 nanoparticles must be embellished to some extent for further use in drug delivery. This problem can be solved by modifying the structure of Fe3O4 nanoparticles with a thick carbon layer and abundant functional groups [35–37]. The high porosity and abundant –OH groups provide Fe3O4@C a superior adsorption performance on substances through electrostatic attractions. Notably, Fe3O4@C nanospheres could still be well dispersed in water and easily separated by an external magnet because of their excellent hydrophilic property and superparamagnetism [35,38]. Based on the above background, layer-by-layer (LBL) self-assembly technology [39] was adopted in this experiment to prepare dual-core shell pH-sensitive Fe3O4@C/CMC/CS microspheres. The stability and spheronization of the whole system were greatly improved by adding Fe3O4@C nanospheres to the CMC matrix. Through ionic interactions between carboxylate anions of CMC and cationic amine groups of chitosan, the pH-sensitive carrier for loading and delivery of diclofenac sodium (DS; an anti-inflammatory drug) can be expected to be used for gastrointestinal tract applications of controlled-release drug carrier. The developed pH-sensitive Fe3O4@C/CMC/CS microbeads were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectrophotometry (FTIR), and thermogravimetric analysis (TGA), and the drug loading efficiency and loading content were estimated. The pH sensitivity of the developed Fe3O4@C/CMC/CS microbeads was examined from the point of
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measuring their swelling degree and in vitro drug release under simulated gastrointestinal tract conditions. In brief, the generated composite PECs could be expected to improve the encapsulation efficiency of DS, minimize its loss in stomach, and control its release in a localized colon region. 2. Materials and methods 2.1. Materials Carboxymethyl cellulose (MW = 90,000 (DS = 0.7), viscosity: 50–100 mPa.s), chitosan (deacetylation degree N95%, viscosity: 100–200 mPa.s), ferrocene (Fe(C5H5)2, purity of 99%), diclofenac sodium, aluminum chloride (AlCl3), polyethylene glycol (PEG, MW = 1000), hydrogen peroxide (H2O2, purity of 30%), acetone (C3H6O, purity of 99%), disodium hydrogen phosphate, sodium dihydrogen phosphate, and hydrochloric acid were purchased from Aladdin Chemical Co. Ltd. (Shanghai, China). All these reagents and chemicals were of analytical grade. 2.2. Preparation of Fe3O4@C According to the literature [38], Fe3O4@C was synthesized through one-step solvent thermal treatment, and 1:1 was adopted as the optimal proportion of composite. After accurately weighing 1.6 g ferrocene and 1.6 g PEG in a 100-mL volumetric flask, 70 mL of acetone was added and blended well until complete dissolution of ferrocene and PEG. After magnetic stirring (120 rpm) for 30 min, H2O2 (4 mL) was dropped slowly into the solution and continuously stirred for 2 h. Then, the entire solution was transferred from the flask to a 100-mL Teflon-lined stainless-steel autoclave and maintained at high temperature (200 °C) for 48 h. As the vessel cooled, the precipitates were collected via magnetic separation and washed at least three times with alcohol. Finally, the black products (Fe3O4@C) were dried at 60 °C in a vacuum oven for 12 h. 2.3. Preparation of Fe3O4@C/CMC/CS composite beads The DS delivery system was obtained by mixing the prepared CMC solution with Fe3O4@C and DS, followed by coating with chitosan. The formulation composition for preparation of Fe3O4@C/CMC/CS is given in Table 1. Typically, a weighted amount of CMC was dissolved in hot
Scheme 1. Preparation of pH-sensitive Fe3O4@C/CMC/CS composite beads.
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distilled water at 60 °C with stirring for 1 h until a homogenous and uniform solution was obtained. Then, a predetermined amount of Fe3O4@C was dispersed in distilled water and added slowly to the above CMC aqueous solution. The viscous liquid was prepared by ultrasonic dispersing technology for 1 h to obtain a uniform dispersion system. Subsequently, the suspension containing CMC and Fe3O4@C was extruded in the form of droplets using a 5-mL syringe (inside diameter of around 10 mm) into 100 mL of a certain concentration of AlCl3 solution at an invariable dropping rate at 25 °C. The formed spherical Fe3O4@C/CMC beads were
allowed to crosslink for 1 h under slow stirring to solidify the core, followed by rinsing with distilled water to remove unreacted AlCl3 on the surface of the beads. Finally, the semi-products were immersed in a certain concentration of chitosan solution for 2 h, resulting in instantaneous formation of core-shell beads due to electrostatic interactions between chitosan and CMC. These coated beads were washed with distilled water to remove excess of unreacted chitosan and dried in a vacuum oven at 40 °C for further use. The presumptive mechanistic route for the formulation of Fe3O4@C/CMC/CS beads is shown in Scheme 1.
Fig. 1. Scanning electron microscopy (SEM) images of Fe3O4@C (a), Fe3O4@C/CMC/CS beads (b), Fe3O4@C/CMC (c) and its larger image (d), Fe3O4@C/CMC/CS powder (e) and its larger image (f).
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Fig. 2. Transmission electron microscopy (TEM) images of Fe3O4@C (a), Fe3O4@C/CMC (b), Fe3O4@C/CMC/CS powder (c), and Fe3O4@C/CMC/CS beads (d).
The preparation of drug-loading Fe3O4@C/CMC/CS beads was the same as above. A certain concentration (1 mg/mL) of DS solution was transferred into the mixture in the same way to obtain a final volume of 100 mL.
predetermined time intervals, the excess surface-adhered liquid drops were removed by blotting with a filter paper, and the swollen beads were weighed again immediately. The swelling ratios of the microcapsules were calculated using Eq. (1):
2.4. Characterization Swelling ratio ð%Þ ¼ The surface morphology of the microgel beads was studied with a SEM (S-4800, Hitachi Limited Ltd., Japan). For SEM, all samples were coated with a thin gold film under vacuum prior to microscopy analysis, and these samples were viewed using an accelerating voltage of 15 kV at the appropriate magnification. TEM images of Fe3O4@C powder, Fe3O4@C/CMC, and Fe3O4@ C/CMC/CS beads were obtained using Lorentz Transmission Electron Microscope (TEM, JEM-2100, JOEL, Japan). FT-IR (Tensor II, Bruker, Germany) was used to characterize the functional groups of the materials. In each case, the powder samples were placed into KBr (spectrum pure) pellet, and then the spectra were scanned and recorded from 400 to 4000 cm−1 at room temperature (25 °C). Furthermore, TGA of all samples was performed by employing a TGA (Pyris Diamond TG/DTA, Perkin-Elmer, USA) at a heating rate of 20 °C/min from 20 to 800 °C under N2 atmosphere.
W t −W 0 100% W0
ð1Þ
where Wt is weight of the swollen microcapsules at time t and W0 is the initial weight of microcapsules.
2.5. Swelling ratio studies The swelling behavior was studied by measuring the percentage of water uptake by microcapsule beads via the immersing method. Accurately weighed 0.1 g of dried beads were allowed to swell in 50 mL of simulated gastric fluid (SGF, pH 1.2), simulated intestinal fluid (SIF, pH 6.8), and simulated colonic fluid (SCF, pH 7.4) at 37 °C. At
Fig. 3. FT-IR spectra of (a) CMC, (b) chitosan, (c) Fe3O4@C, (d) Fe3O4@C/CMC, and (e) Fe3O4@C/CMC/CS.
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where We is the amount of drug in the weighted amount of microcapsules in equilibrium and W0 is the amount of drug that was first added during preparation procedure. 2.7. In vitro drug release studies For in vitro DS release studies from the loaded microcapsules, three different pH solutions (SGF, SIF, and SCF) were investigated. In each experiment, 0.1 g of dried microcapsule beads were immersed in SGF (2 h) and then transferred to SIF (4 h) or SCF (4 h) at 37 °C in a water bath at 100 rpm to ensure that all the encapsulated DS was extracted by the used pH buffer. At predetermined time intervals, approximately 3 mL of the buffer solution was removed and filtered for assaying at 284 nm using an UV spectrophotometer. The removed solution was replaced with the same volume of fresh PBS. The amount of drug released from the beads was determined by using a calibration plot curve obtained under the same conditions. Fig. 4. TGA of CMC (a), Fe3O4@C (b), Fe3O4@C/CMC (c), and Fe3O4@C/CMC/CS beads (d).
3. Results and discussion
2.6. Drug loading studies
3.1. Analysis of shape and appearance
The loading of DS into all the microcapsule beads was achieved by the wrapped method as mentioned above (preparation of microcapsules). Then, the dried drug-loaded beads were crushed in a mortar with a pestle, followed by sifting of the polymeric powder (0.1 g) to an Erlenmeyer flask using a 200-mesh sieve. Subsequently, 50 mL (pH 7.4) of phosphate-buffered solution (PBS) was added with shaking at 25 °C for 24 h in a water bath to ensure complete extraction of DS from the beads. After 1 h, precipitated gels were filtered; DS was analyzed by using a UV spectrophotometer at a wavelength of 284 nm using a calibration curve and PBS (pH 7.4) as the blank. The percentage of entrapment efficiency was then calculated using Eq. (2):
To analyze Fe3O4@C/CMC/CS beads and determine the shape and appearance of the constituent components further, Fe3O4@C, Fe3O4@ C/CMC, and Fe3O4@C/CMC/CS powders were examined respectively by SEM and TEM, as shown in Figs. 1 and 2. As shown in Scheme 1 and Fig. 1, magnetic iron oxide particle (Fig. 1a), which was surrounded with PEG, showed more centralized magnetism. The wet beads (Fe3O4@C/CMC/CS) were almost spherical and smooth, with an average particle size of approximately 3 mm and gray color (that changed slowly to black with increasing amount of Fe3O4@C). The particle size of composite microspheres when dried at 40 °C tended to become smaller to approximately 1.3 mm. The surface of microspheres became rough and porous (Fig. 1b). This could be because the process of drying led to partial collapse of the polymeric gel network structure.
Drug encapsulation efficiencyð%Þ ¼
We 100% W0
ð2Þ
Fig. 5. Effect of Fe3O4@C concentration (a), CMC concentration (b), AlCl3 concentration (c), and chitosan concentration (d) on DS encapsulation efficiency of Fe3O4@C/CMC/CS beads.
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Fig. 6. Effects of Fe3O4@C concentrations on the swelling ratio of Fe3O4@C/CMC/CS beads in SGF (a), SIF (b), and SCF (c). Effects of CMC concentrations on the swelling ratio of Fe3O4@ C/CMC/CS beads in SGF (d), SIF (e), and SCF (f).
Comparison of the SEM images of Fe3O4@C/CMC (Fig. 1c) and Fe3O4@C/CMC/CS (Fig. 1e) showed that the surface of Fe3O4@ C/CMC/CS beads was less wrinkled. This was attributed to the assembly of chitosan on the Fe3O4@C/CMC layer to form a neat, tight, and flat surface by interactions between CMC and chitosan. In addition, the measurement of diameter of Fe3O4@C/CMC/CS wet bead constricts during the chitosan coating process was an acknowledgement of the interaction. It can be seen from Fig. 1d that Fe3O4@C particles were wrapped into the CMC colloid. A magnified version of Fe3O4@C/CMC/CS (Fig. 1f) after the assembly of chitosan on the Fe3O4@C/CMC layer showed that the surface of composite microspheres had numerous wrinkled areas, which favored water and tissue fluid infiltration. TEM was used to determine the crystallographic and morphological discrepancy of these as-prepared beads with high spatial resolution (Fig. 2a–d). The Fe3O4@C inner cores (Fig. 2a) were magnetic black
globules that were sealed uniformly with PEG. When the inner cores were wrapped into CMC to solidify in AlCl3 solution, the magnetic black globule attracted each other to form a more compact and stable sphere (Fig. 2b). As shown in Fig. 2c, after coating with chitosan, the interfacial interactions caused shrinking of the layers to form a more stable system. 3.2. FT-IR FT-IR analysis of raw CMC, chitosan, Fe3O4@C, Fe3O4@C/CMC, and Fe3O4@C/CMC/CS beads was carried out, and the data are shown in Fig. 3. FT-IR analysis was performed to verify that CMC and chitosan were coated on the system during material preparation. In the spectrogram of raw CMC (Fig. 3a) and chitosan (Fig. 3b), the peaks at 3445 cm−1 and 3430 cm−1 were respectively caused by the stretching
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Fig. 7. Effects of AlCl3 concentrations on the swelling ratio of Fe3O4@C/CMC/CS beads in SGF (a), SIF (b), and SCF (c). Effects of chitosan concentrations on the swelling ratio of Fe3O4@ C/CMC/CS beads in SGF (d), SIF (e), and SCF (f).
vibration of O\\H in CMC, and O\\H and N\\H in chitosan. A broad asymmetrical band was observed at 1645 cm−1 and a narrower symmetrical band at 1420 cm−1, which corresponded to the basic characteristic functional group –COO−. In the chitosan spectrum, the absorption bands at 1640 cm−1 corresponded to carbonyl stretching of the secondary amide. The bands between 1300 and 1460 cm−1, and at 1000–1200 cm−1 are assigned to C\\N and C\\O stretching on the polysaccharide skeleton, respectively [40]. Obvious changes were observed in the FT-IR spectrum of Fe3O4@ C/CMC/CS core–shell beads compared with those of the other samples. In the Fe3O4@C/CMC/CS microspheres, movement to a lower frequency of 3425 cm−1 indicated the formation of hydrogen bonds between the carboxyl group of CMC and the hydroxyl group of chitosan, as well as the superposition of N\\H and O\\H groups. The line (c) demonstrates a peak at 596 cm−1, which is attributed to Fe\\O
bond [41]. The bands at 1638 cm−1 were attributed to –C_O bond. These bands combined with the presence of the stretching vibration of O\\H illustrated that the carbon layer and abundant functional groups were wrapped into Fe3O4 magnetic nanoparticles [42]. As can be seen in Fig. 3d–e, a new absorption band was observed at 1480 cm−1, which can be interpreted in successfully coated chitosan layer. Some small peaks increased in strength between 1400 cm−1 and 1600 cm−1, which indicated the form of PEC structure. 3.3. TGA Thermogravimetric curves determined for CMC, Fe3O4@C, Fe3O4@ C/CMC, and Fe 3 O4 @C/CMC/CS microcapsule beads are shown in Fig. 4. From the degradative curves of Fe3O4@C (Fig. 4b), it was evident that the magnetic iron oxide was successfully wrapped into
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PEG. Since –OH and –CH of Fe3O4@C were carbonized, the weight of the sample was reduced slightly when the temperature increased. Similarly, the weight curves of Fe3O4@C/CMC (Fig. 4c) and Fe3O4@ C/CMC/CS (Fig. 4d) showed a gradual decline to 80%. This is due to the degradation of the polymer and hydrogen-bound water in the temperature range of 80–200 °C, which forms the polysaccharide structure of CMC and CS. In addition, a uniform and smooth decline in weight loss at 250–400 °C in Fe 3O 4 @C/CMC and Fe3 O 4@ C/CMC/CS was observed. This may be partly attributed to the degradation and decomposition of organic skeletal structure, amino groups, and other functional groups. By comparing the curves of Fe3O4@C/CMC and CMC (Fig. 4a), it was observed that Fe3O4@C particles are wrapped into CMC and can enhance the thermal stability of the whole system. At a temperature of 500 °C or higher, the remaining material was carbonized completely. Comparison of the degradation processes of Fe3O4@C/CMC and Fe3O4@C/CMC/CS showed that the weight loss rate for the latter was much higher than that for the former at the same condition. This indicated that chitosan coated successfully and penetrated deeply into the surface of Fe3O4@C/CMC by forming a PEC through polar interactions between carboxylate groups (CMC) and amino groups (chitosan). In addition, Fe3 O 4 @ C/CMC/CS composite beads were more thermally stable than Fe 3 O4 @C/CMC. Given the above, it can be suggested that the Fe3O4@C/CMC/CS drug delivery system would be thermally stable at the physiological temperature of the human body. 3.4. DS encapsulation efficiency Drug loading efficiency is a crucial parameter to determine the effectiveness of drug delivery vehicle [43,44]. Excellent drug loading rates may effectively increase the application of drug-carrying systems and reduce the waste of bulk drug substances. In the present study, we
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investigated the encapsulation efficiency of DS in diverse Fe3O4@ C/CMC/CS beads by comparing the effects of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan. In the experiment, the initial concentration of DS was constant and equivalent (1 g/L). The percentage of DS encapsulation was around 55–80% and it varied with the concentration of raw materials as shown in Fig. 5. The encapsulation of hydrophilic molecules such as DS remains a significant challenge, because these drugs exhibit a strong tendency to leak to the external aqueous phase. The factors that affect the encapsulation of hydrophilic molecules include concentrations of matrix materials and crosslinker during encapsulation process, indicating that Fe3O4@C/CMC/CS system can be an excellent drug carrier. DS was successfully loaded into Fe3O4@C/CMC/CS beads and the encapsulation efficiency (%) increased from 52.2 ± 0.79% to 70.8 ± 0.65% with increase in concentration of Fe3O4@C from 0.025% to 0.1% (CMC: 3%; AlCl3: 3%; CS: 1%), as shown in Fig. 5a. This illustrates the importance of Fe3O4@C particles in the whole system, because they showed concentrated magnetism and formed a steady inner core structure. However, if the concentration of Fe3O4@C (i.e. 0.2%) is increased continuously, the microsphere structure will show a tendency to burst and rupture. This could be because of the overweight of Fe3O4@C particles and magnetic interaction. The compound beads were hard to solidify when the concentration of CMC was low (i.e. 1–2%). The colloid with excessive levels (exceeds 5%) of CMC was too thick to form droplets; therefore, concentrations of CMC from 3%–5% were adopted to analyze the effect of CMC concentration. The results (Fig. 5b) showed that the drug loading efficiency of 3% CMC reached a peak (70.8 ± 0.65%), and by increasing the concentration of CMC to 4% and 5%, the encapsulation efficiency (%) reached 69.53 ± 1.10% and 65.93 ± 0.70%, respectively. A possible explanation for this profile is that the thick matrix has a more compact structure and is easy to crosslink in AlCl3 solution. However, when the concentration of CMC was increased, the dissolution and swelling of CMC had an effect on the solubility of DS. Thus, the encapsulation
Fig. 8. Effects of Fe3O4@C concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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efficacy (%) showed a slightly downward trend and the gel turned turbid in the preparation process. Taken together, the optimum concentration of CMC as the gel stroma was 3%, considering the encapsulation efficacy (%), homogeneity of dosage, and economic factors. Regarding the effect of cross-linking agent (AlCl3) concentration, the rate of interaction of CMC beads and AlCl3 solution increased with increase in concentration of cross-linking agent, leading to a lesser chance of the drug oozing out. When the AlCl3 concentration increased from 2% to 4%, the encapsulation efficacy (%) increased from 62.5 ± 0.52 to 75.1 ± 0.82, as shown in Fig. 5c. Nevertheless, the encapsulation efficacy (%) did not continue to increase, despite the increase in AlCl3 concentration. This could be attributed to the fact that the overall surface area of CMC beads remained unchanged. Finally, when increasing the CS concentration from 0.5% to 1.5% (Fig. 5d), the loading efficiency increased from 62.6 ± 0.94% to 77.5 ± 0.76%. This indicates that the NH3+ group of chitosan exhibited electrostatic interactions with the –COO– group of CMC during PEC formation process, and the thicker chitosan layer (1.5%) resulted in entrapment of drugs in the more thicker PEC shell, which was better than paper shell beads. It is also reasonable to assume that the higher the CS concentration, the thicker the crust of beads and lower the drug loss during the preparation process (long contact and more washing time during the coating process). Similarly, the limited surface groups led to restricted encapsulation efficacy (%). In summary, the concentration of Fe3O4@C, CMC, AlCl3, and chitosan has an effect on the association of DS to Fe3O4@C/CMC/CS beads. The encapsulation efficiency of DS in Fe3O4@C/CMC/CS beads was more balanced (70.8%) at 0.1% Fe3O4@C, 3% CMC, 3% AlCl3, and 1% chitosan. 3.5. Swelling studies Swelling has been considered one of the most important properties of bio-nanocomposite hydrogel beads that determine the release rate of
the encapsulated drug [45,46]. It indicates the easiness and speed of a liquid to penetrate the beads and affect drug diffusion. The swelling degree is used to determine the ability of hydrogels to soak up water, which can be quantitatively analyzed by increasing the size and weight of beads. To investigate this further, we must consider the effects of factors such as solubility of polymers, porosity, nature of ionic groups, and formation of ionic bond. As shown in Figs. 6 and 7, the effects of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan on the swelling behavior of Fe3O4@C/CMC/CS beads were determined at pH of 1.2, 6.8, and 7.4. The swelling behavior was dependent on the following factors: (i) pH, (ii) time, and (iii) concentrations of raw materials. Swelling increased and the growth rate decreased with time, and then gradually reached the maxima. Some beads began to disintegrate. All investigated Fe3O4@C/CMC/CS beads with different contents maintained their structures in acidic environment (SGF). However, with increase in pH, the swelling rate increased by dozens of times (1000–3000%) and their structures were destroyed. This pH-dependent swelling behavior can be explained by the maintenance of CMC–Al3+ complexes in microcapsule core. In addition, only a few chitosan amine groups were protonated in the exterior shell in SGF. The increased pH induced deprotonation of ionizable carboxylic groups on the CMC chain. Moreover, phosphate ions displaced Al ions within the beads after the shell was destroyed, leading to higher electrostatic repulsion between these charged groups. This subsequently causes an increase in osmotic pressure and amount of water penetrating into the molecules. Our study discusses the effect of different concentrations of Fe3O4@C particles, and the results are shown in Fig. 6a–c. Because of the frangibility and instability of pure CMC/chitosan beads, the experiment was conducted without regard to pure beads. At lower concentrations of Fe3O4@ C particles, the moisture-absorbing rate of Fe3O4@C/CMC/CS beads was faster, swelling ratio was lower, and the whole system collapsed rapidly due to inadequate material strength. With the increase in Fe3O4@C
Fig. 9. Effects of CMC concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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particles, Fe3O4@C/CMC/CS beads adsorbed more water and maintained the form with no changes in swelling state. A possible explanation for this is that Fe3O4@C particles could support the hydrogel network and enhance the mechanical strength of CMC matrix, which are important in drug loading. Notably, exorbitant concentrations of Fe3O4@C particles can result in overweight load and excessive magnetism, which in turn can lead to collapse of the whole system. This underscored the significance and indispensability of appropriate Fe3O4@C particles. Because low CMC concentrations (i.e. 1%, 2%) were too sparse to maintain the structure, we selected 3%–5% CMC concentrations to study the effect. Based on the data presented in Fig. 6d–f, CMC concentrations of 4% and 5% resulted in the highest swelling rates at pH 6.8 and 7.4, indicating that the concentration of CMC as the matrix played an important role in swelling performance. This is due to the reaction balance between –COO−, Al ions, and –NH2 at lower CMC concentrations, and the saturation of chelation between Al3+ and –COO– led to the formation of a dense nucleus. As the CMC concentration increased, an external layer was formed by PECs, wherein the amine groups of chitosan electrostatically interacted with a much larger number of carboxyl groups of CMC. As shown in Fig. 6d, under acidic conditions, 3% CMC beads were more easily swollen and maintained their structure and morphology in 6–8 h. The more the CMC chain was exposed, the more the amount of infiltrated water and the easier the disintegration of the swelling core. Based on these results, we speculated that increased CMC concentration leads to stronger water absorption capacity and a tighter and engorged inner core when other conditions are immobilized. As shown in Fig. 7a–c, under acidic conditions, the swelling ratio reached the peak rapidly (1450%) at lower concentration of AlCl3 (2%). In contrast, with increase in AlCl3 concentration, the swelling ratio tended to reduce. This could be because of rapid saturation of chelation
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between Al3+ and –COO−, subsequently resulting in the formation of a tough nucleus making it hard for water to penetrate. At 4% AlCl3 concentration, the site of bead surface had reached saturation, and predictably, higher AlCl3 concentration (N4%) only caused a slight increase. When the pH reached 6.8, a major increase in swelling ratio was observed. The penetrative process led to a slow swelling trend with increase of AlCl3 concentration, and the inner core structure became more stable. Similar trends were observed when the pH reached 7.4. However, the swelling ratio changed according to chitosan content (Fig. 7d–f). The inner core (CMC-Al3+ interface) tended to shrink at lower pH condition; thus, the outermost chitosan layer plays a major role in preventing water infiltration during the swelling process. The swelling capacity improved with increase in chitosan concentration from 0.5% to 1.5% at pH 6.8 and 7.4. This might be due to increased protonation of amine groups with increase in amount of –NH2 and decreased protonation with increase of pH. As the CS concentration increased, the swelling degree also increased, suggesting that the interaction between CMC and chitosan lines was reinforced. The intensive interaction is easy to cause an increase in osmotic pressure. Therefore, the swelling ratio of 1.0–1.5% chitosan ultimately attained a higher level at pH 6.8 and 7.4. Based on these results, the prepared Fe3O4@C/CMC/CS composite hydrogel beads can be applied for sustained drug delivery under different pH conditions. 3.6. Release studies A favorable drug control-release system is expected to retain enough amount of drug in the stomach and to release more amount of the drug in the intestine [47]. Therefore, orally administered drug-loaded polymeric formulations must have excellent pH sensitivity. However, a
Fig. 10. Effects of AlCl3 concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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sudden release of drug in the initial phase is not expected. To study the effects of Fe3O4@C, CMC, AlCl3, and chitosan concentrations on the release of DS from Fe3O4@C/CMC/CS composite beads, accurately weighed quantity (0.1 g) of dry composite beads were immersed in SGF (2 h) and transferred to SIF (4 h) or SCF (4 h) at 37 °C, and their cumulative release ratio was analyzed. The cumulative release ratio always maintained an extremely low value under acidic conditions (Fig. 8c). Within the error range allowed, there was a slight fluctuation in the release ratio of three different Fe3O4@C concentrations. After switching to the neutral (Fig. 8b) and alkaline (Fig. 8c) systems, the cumulative release ratio increased markedly, and higher concentration of Fe3O4@C corresponded to lower release. A possible explanation for this might be that the structure of the beads remained stable at higher Fe3O4@C concentrations leading to slower process of release and disintegration, mainly dependent on the magnetic properties. The cumulative release data shown in Fig. 9 indicated that as the concentration of CMC increased from 3% to 5%, a considerable decrease in DS release was observed at pH 1.2, 6.8, and 7.4. This result was attributed to the shrinking of CMC gel. More number of carboxyl groups of CMC are in the unionized form for sustained drug release in the stomach. To a certain extent, the higher the CMC concentration, the more compact the inner core, resulting in harder disintegration and decreased release ratio in phosphate buffer at pH 6.8 and 7.4. As shown in Fig. 10, when the AlCl3 concentration increased from 2% to 4%, the total amount of the released DS decreased in SGF, SIF, and SCF. The result showed that the drug release was higher at lower AlCl 3 concentration, because the more the number of Al3+ ions around CMC, the faster the formation of a tight inner core. The rigorous surface was caused by the complexation between Al 3+ and the hydroxyl and carboxyl groups of CMC, resulting in blockade of drug release. This finding is consistent with the previously
described swelling profile of Fe3O4@C/CMC/CS. In addition, we predicted that with the increase in AlCl3 concentration, a similar continuous decline in release ratio may not be observed because of the limited surface area. Finally, we also analyzed the effect of chitosan concentrations on drug release (Fig. 11). In acidic conditions, the degree of swelling and the protonation of amino groups of chitosan increased, resulting in easy release of drug from beads. The noticeable changes observed in SIF and SCF can be explained by the ionic interactions between the carboxyl groups of CMC and ammonium groups of chitosan by forming PEC, whose density increases with increasing concentrations of the polymers. In addition, the electrostatic interaction of drug-containing systems is easily broken at pH 6.8 and 7.4 than at pH 1.2. However, a shrinking tendency of the outer layer of chitosan was observed at pH 6.8 and 7.4, which ensured that the whole system remained in steady state for a period of time after increased water absorption and swelling of internal structure. Overall, the amount of DS released from Fe3O4@C/CMC/CS beads within 2 h was low (b10%) at pH 1.2. When the pH was changed from near neutral to alkaline, the eventual release rate showed a significant increase. The extent of protonation, properties of the model drug, swelling ratio, and drug release are all interlinked. A possible explanation might be that the microspheres began to swell after entering the liquid environment and external amino groups of chitosan are gradually ionized. Internal Fe3O4@C and CMC layer make the inner core tighter for preventing the entry of water molecules into the nucleus and for allowing the drug to leach out of the wet beads for longer periods. In the near neutral and alkaline phosphate buffer, the networks became more hydrophilic because of the formation of neutralized carboxyl in the CMC layer, leading to a collapsed state. Particularly, the release ratio was still maintained at 40–50% in the initial neutral and alkaline stages. These results reveal that the whole Fe3O4@C/CMC/CS composite
Fig. 11. Effects of chitosan concentrations on DS release profiles of Fe3O4@C/CMC/CS beads in SGF-SIF (a), SGF-SCF (b), and SGF (c).
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system can be an excellent controlled-release drug carrier in the pHsensitive environment of the gastrointestinal tract. 4. Conclusion Fe3O4@C particles serve as promising supporting materials for pHsensitive composite beads of Fe3O4@C/CMC/CS. The synthesized composite beads were characterized by SEM, TEM, FT-IR, and TGA. In addition, the drug encapsulation efficiency, pH-responsive swelling, and drug release behavior of the Fe3O4@C/CMC/CS system were studied based on the composition of the formulations, including the effect of different concentrations of Fe3O4@C, CMC, AlCl3, and chitosan. The swelling and release depended on the pH, time of incubation, and concentrations of raw materials. The beads showed a higher swelling ratio and release in phosphate buffer at pH 6.8 and 7.4 than at pH 1.2, and prevented a sudden release. These results indicated that Fe3O4@ C/CMC/CS composite beads in proper proportion can be an excellent and directed controlled-release drug carrier. Acknowledgments This work was financially supported by the National Natural Science Foundation of China (21476212) and Bureau of Science and Technology of Zhoushan (2017C41005). References [1] K.S. Soppimath, T.M. Aminabhavi, A.R. Kulkarni, W.E. Rudzinski, Biodegradable polymeric nanoparticles as drug delivery devices, J. Control. Release 70 (1) (2001) 1–20. [2] R. Masteiková, Z. Chalupová, Z. Sklubalová, Stimuli-sensitive hydrogels in controlled and sustained drug delivery, Medicina 39 (Suppl. 2) (2003) 19. [3] C. Isha, S. Nimrata, A.C. Rana, G. Surbhi, Oral sustained release drug delivery system: an overview, Int. Res. J. Pharm. 3 (5) (2012). [4] K.Y. Lee, S.H. Yuk, Polymeric protein delivery systems, Prog. Polym. Sci. 32 (7) (2007) 669–697. [5] G. Meera, A.T. Emilia, Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan–a review, J. Control. Release Off. J. Control. Release Soc. 114 (1) (2006) 1–14. [6] A.P. Rokhade, S.A. Agnihotri, S.A. Patil, N.N. Mallikarjuna, P.V. Kulkarni, T.M. Aminabhavi, Semi-interpenetrating polymer network microspheres of gelatin and sodium carboxymethyl cellulose for controlled release of ketorolac tromethamine ☆, Carbohydr. Polym. 65 (3) (2006) 243–252. [7] T. Coviello, P. Matricardi, C. Marianecci, F. Alhaique, Polysaccharide hydrogels for modified release formulations, J. Control. Release 119 (1) (2007) 5–24. [8] P. Bhusal, J. Harrison, M. Sharma, D.S. Jones, A.G. Hill, D. Svirskis, Controlled release drug delivery systems to improve post-operative pharmacotherapy, Drug Deliv. Transl. Res. 6 (5) (2016) 1–11. [9] J. Zhang, Q. Wang, A. Wang, In situ generation of sodium alginate/hydroxyapatite nanocomposite beads as drug-controlled release matrices, Acta Biomater. 6 (2) (2010) 445–454. [10] M. Kissel, P. Peschke, V. Subr, K. Ulbrich, J. Schuhmacher, J. Debus, E. Friedrich, Synthetic macromolecular drug carriers: biodistribution of poly[(N-2-hydroxypropyl) methacrylamide] copolymers and their accumulation in solid rat tumors, PDA J. Pharm. Sci. Technol. 55 (3) (2001) 191–201. [11] M. Kissel, P. Peschke, V. Šubr, K. Ulbrich, A.M. Strunz, R. Kühnlein, J. Debus, E. Friedrich, Detection and cellular localisation of the synthetic soluble macromolecular drug carrier pHPMA, Eur. J. Nucl. Med. Mol. Imaging 29 (8) (2002) 1055–1062. [12] X. Li, M.A. Kanjwal, L. Lin, I.S. Chronakis, Electrospun polyvinyl-alcohol nanofibers as oral fast-dissolving delivery system of caffeine and riboflavin, Colloids Surf. B: Biointerfaces 103 (2) (2013) 182–188. [13] A. Alves, S.G. Caridade, J.F. Mano, A.S. Rui, L.R. Rui, Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae, Carbohydr. Res. 345 (15) (2010) 2194–2200. [14] S. Jana, A. Gandhi, K.K. Sen, S.K. Basu, Natural polymers and their application in drug delivery and biomedical field, J. PharmaSciTech 1 (1) (2011) 16–27. [15] A.J. Smit, Medicinal and pharmaceutical uses of seaweed natural products: a review, J. Appl. Phycol. 16 (4) (2004) 245–262. [16] D.A. Giovanna Gomez, M. Mario, L. Paola, Marine derived polysaccharides for biomedical applications: chemical modification approaches, Molecules 13 (9) (2008) 2069–2106. [17] A. Anabela, S.G. Caridade, J.O.F. Mano, A. Rui Sousa, L. Rui Reis, Extraction and physico-chemical characterization of a versatile biodegradable polysaccharide obtained from green algae, Carbohydr. Res. 345 (15) (2010) 2194–2200. [18] M.S.M. Eldin, H.M. El-Sherif, E.A. Soliman, A.A. Elzatahry, A.M. Omer, Polyacrylamide-grafted carboxymethyl cellulose: smart pH-sensitive hydrogel for protein concentration, J. Appl. Polym. Sci. 122 (1) (2011) 469–479.
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