Preparation of magnetic pH-sensitive microcapsules with an alginate base as colon specific drug delivery systems through an entirely green route

Abstract

The aim of this work was to prepare pH-sensitive drug carriers for colon specific drug delivery through a completely green and environmentally friendly route (without using any organic solvents, hazardous chemicals and even a harsh procedure). To achieve this, natural biopolymers (sodium alginate, chitosan and carboxymethylcellulose sodium) were used. Naproxen (as a model drug) and magnetic nanoparticles were encapsulated into the alginate microcapsules by formation of an alginate–Ca²⁺ complex. To overcome the drawbacks of the alginate microcapsules like porosity and burst drug release, they were coated with chitosan and carboxymethylcellulose sodium by a layer-by-layer technique that led to the formation of polyelectrolyte complexes through inter-ionic interactions between oppositely charged biopolymers. After coating with these natural polymers, porosity was reduced and burst drug releases were modified. The prepared magnetic microcapsules were characterized by FT-IR, DSC and SEM to study their structures and the roles that each polymeric layer plays. The swelling ratios and in vitro drug release profiles of the microcapsules were studied in both enzyme-free simulated gastric (SGF, pH 1) and simulated colonic fluids (SCF, pH 7.4). These microcapsules could be driven by means of an external magnet and also the results show that these coated microcapsules are completely pH-sensitive and potentially can be used as stimuli responsive carriers for colon specific drug delivery.

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1. Introduction

Although the oral route is one of the most desirable and popular approaches for drug administration, for some drugs the oral route shows many shortcomings. For example, these drugs are degraded and decomposed by the severely acidic medium of the stomach or by enzymatic attack in the upper small intestinal tract. To date, several approaches have been reported to overcome these problems. In recent years, controlled release systems have been showing significant potential in the medical and pharmaceutical fields. These systems are designed to improve drug therapies, in other words, they reduce the side effects and increase the desired effects of drugs.^1–9 Many papers have been published about the appropriate materials and routes to prepare controlled drug delivery systems, but unfortunately some of them used unsafe materials or harsh procedures.^10–14 One class of materials that widely used in drug delivery systems is pH-sensitive polyelectrolyte hydrogels. These hydrogels have been recommended for controlled release into the gastrointestinal tract due to their considerable ability to alter solute diffusion rate and swelling behavior, in a response to the different environmental pH.^15–18

Since natural polymers are environmentally friendly materials, hydrogels made from them are preferred to other materials because of their merits (such as availability, low cost, non-toxicity, biocompatibility, biodegradability, etc.).^19–26 Among natural polymers, sodium alginate (Alg) and chitosan (Cs) are widely used in controlled drug delivery systems. Alg, an anionic polysaccharide, is mostly extracted from brown algae. This water-soluble biopolymer consists of two monomeric units (Scheme 1a): alpha-(1-4)-L-guluronic acid (G) and beta-(1-4)-D-mannuronic acid (M). As mentioned before, Alg is soluble in water, but it could form gels in the presence of cross-linking agents (e.g. Ca²⁺, Sr²⁺, Zn²⁺, Mn²⁺, Cu²⁺, Ba²⁺, etc.). These cations are coordinated and form complexes with Alg (Scheme 1b). Although the Ba²⁺ forms a stronger alginate complex than Ca²⁺, the Alg–Ba²⁺ gels are not suitable for pharmaceutical and medical purposes (due to their toxicity).

Scheme 1 Chemical structures of (a) alginate; (b) Alg–Ca²⁺ complex; (c) chitosan and (d) CMC.

Alg gels are stable in acidic medium (SGF) and their swelling in this pH is nominal therefore the drug release would be negligible. In contrast, as the Alg gels reach the gastrointestinal tract (higher pH), they swell and start disintegrating. As a result, the swelling and drug release would be increased. Because of these properties, Alg gels are widely exploited in encapsulation and controlled drug release.^27–36 Nevertheless, natural polymers have some drawbacks such as poor mechanical strength, uncontrolled water uptake, burst release, etc. These problems could be reduced by blending or grafting with other polymers or inorganic materials.^22,37,38 In addition, the chief drawbacks of Alg gels are their porous structure and low physical strength at higher pH media, which can lead to a burst release of encapsulated drugs after they reach the alkaline media (SCF). These limitations can be modulated through applying the Layer-by-Layer (LbL) technique, which leads to the formation of an extra outer layer. It is happened by the formation of polyelectrolyte complexes (PEC) through inter-ionic interactions between a negatively charged polymer and cationic polymers.^39–43 In general, inter-ionic interactions and LbL techniques can be carried out in environmentally friendly conditions (i.e. in aqueous media under mild conditions without using toxic solvents and hazardous chemical cross-linkers), thus using this method could be more convenient than other approaches.^44–49 Numerous studies have been conducted on the treating of Alg microcapsules with Cs. Although Cs modifies the Alg drawbacks (i.e. their porous structure and low physical strength at higher pH media), it has been noticed that these modified Alg microcapsules (Alg/Cs) would be a bit destabilized in lower pH media and consequently a partial amount of encapsulated drug would be released in simulated gastric fluid (SGF).^46,50 Additional polymeric layers can be employed to protect the Alg/Cs microcapsules from destabilization and drug release in the acidic media that in this work carboxymethylcellulose sodium (CMC), an anionic natural polymer, was used for this purpose. Due to its anionic feature, this polymer is appropriate for covering and protecting of Alg/Cs microcapsules (owing to formation of PEC between Cs and CMC).^51,52

Briefly, in this work, magnetic microcapsules with alginate base were prepared under entirely green and environmentally friendly conditions that they could be driven by means of an external magnet. These Alg microcapsules were coated and modified with natural polymers (Cs and CMC) and analyzed by DSC, FT-IR and SEM. Naproxen as a model drug was placed into the microcapsules and the swelling and in vitro release profiles were established in both enzyme-free simulated gastric (SGF, pH 1) and simulated colonic fluids (SCF, pH 7.4) at 37 ± 1 °C.

2. Materials and methods

2.1. Materials

Chitosan (deacetylation degree > 90%), sodium alginate (Alg) and carboxymethylcellulose sodium (CMC) were purchased from Sigma-Aldrich Chemical Co. Calcium chloride (CaCl₂), ammonium hydroxide (NH₄OH), sodium hydroxide (NaOH), iron(II) chloride tetrahydrate (FeCl₂·4H₂O) and iron(III) chloride hexahydrate (FeCl₃·6H₂O) were obtained from Merck Co. All chemicals and reagents were analytical grade and used as received.

2.2. Preparation of Fe₃O₄ nanoparticles (MNPs)

Fe₃O₄ nanoparticles were prepared according to the chemical co-precipitation method. Briefly, FeCl₂·4H₂O (1.72 g) and FeCl₃·6H₂O (4.72 g) were dissolved in deionized water and the mixture was stirred vigorously (900 rpm) under the nitrogen atmosphere. As the mixture was heated to 85 °C, 10 mL of ammonium hydroxide was added dropwise. The solution became dark after addition of ammonium hydroxide. The reaction was refluxed at 80 °C for 60 min. After cooling down, the black precipitate was separated by means of a permanent magnet and washed several times with distilled water and ethanol to remove the unreacted reagents followed by drying at room temperature under vacuum.

2.3. Preparation of magnetic alginate microcapsules (M.Alg)

To prepare magnetic alginate microcapsules (M.Alg), 2 g of Alg was dissolved in deionized water (100 mL). 0.2 g of Fe₃O₄ nanoparticles were dispersed in a water–ethanol solution (consisting of 5 mL of ethanol and 7 mL of deionized water) by ultrasonication and then dropped into the Alg solution under vigorous stirring. 0.4 g of the model drug, naproxen, was added into the mixture. The obtained mixture was ultrasonicated for 1 h in order to prepare well-disperse and bubble-free mixture. The well-dispersed mixture was dropped into the aqueous solution of calcium chloride (2% w/v) through a 0.3 mm syringe needle at a constant dropping rate. The microcapsules were immersed in the calcium chloride solution in the absence of permanent magnet at room temperature for 30 min. Then formed spherical beads were separated by permanent magnet (after hardening) and washed with deionized water (step 1). They were dried at room temperature overnight and stored at 10 °C.

2.4. Preparation of magnetic chitosan coated microcapsules (M.Alg/Cs)

To cover M.Alg microcapsules with Cs layer, ionic interactions between –COO⁻ groups of Alg and –NH₃⁺ groups of Cs took place and polyelectrolyte complexes (PEC) between Alg and Cs were formed. To prepare chitosan coated microcapsules (M.Alg/Cs), the Cs solution (2% w/v) was prepared by dissolving of Cs in acetic acidic solution (5.5 > pH > 5). The wet microcapsules obtained from step 1 were suspended in Cs solution for 30–40 min (step 2). M.Alg/Cs microcapsules were separated and rinsed with deionized water. The wet microcapsules were dried at room temperature for 48 h and kept at 10 °C.

2.5. Preparation of magnetic carboxymethylcellulose/chitosan coated microcapsules (M.Alg/Cs/CMC)

To coat M.Alg/Cs microcapsules with CMC layer, interactions between –COO⁻ groups of Alg and –NH₃⁺ groups of Cs and also between –COO⁻ groups of CMC and –NH₃⁺ groups of Cs were happened and then polyelectrolyte complexes (PEC) between Alg, Cs and CMC were formed. To prepare CMC solution, CMC was dissolved in warm deionized water, then the wet microcapsules obtained from step 2 were transferred into the CMC solution and stirred for 40 min (step 3). All microcapsules were separated and followed by washing thoroughly with deionized water then dried at room temperature for 72 h and stored at 10 °C.

2.6. Apparatus measurements

The FT-IR spectra of all microcapsules were recorded using a “Bruker Vector 22” FT-IR spectrometer. Powder samples were placed into the KBr pellet and spectra were recorded at room temperature. The DSC curves were recorded by “Mettler Toledo DSC822e” to investigate the thermal behavior of all materials. The crimped samples were heated in sealed aluminum pans under nitrogen flow (20 mL min⁻¹). A heating rate of 20 °C min⁻¹ was maintained for all samples. Empty aluminum pan was used as a reference. DSC was employed to study thermal properties, the composition of microcapsules and possible interactions between the polymers. Powder X-ray diffraction (XRD) patterns of the samples were recorded on a Bruker AXS model D8 Advance diffractometer using CuKα radiation (λ = 1.542 Å), with the Bragg angle ranging from 2–70°. Morphology and surface characteristics of the samples were studied by SEM images recorded by a Scanning Electron Microscopy (Cam Scan MV 2300).

2.7. Drug encapsulation efficiency

The direct procedure (described elsewhere⁴⁶), was used to extract and then determine the drug content in microcapsules. A partial amount of dried microcapsules were accurately weighed and then crushed (to make drug extraction easy). They were transferred into the phosphate buffer solution (pH 7.4) and stirred at room temperature for 24 h. The solutions were filtered through the nano-filter and then, the drug concentrations were determined spectrophotometrically at the absorption maximum (λ_max) of the drug using a 1 cm quartz cell. The drug encapsulation efficiencies of all microcapsules were calculated by eqn (1):where:

Q_t = the amount of drug that firstly added during preparation procedure;

Q_e = the amount of drug in the accurately weighted amount of microcapsule.

2.8. Swelling study

Swelling characteristics of all magnetic microcapsules were determined via the immersing method, which is used frequently for this purpose.^46,53 The dry and accurately weighed magnetic microcapsules were immersed in simulated gastric fluid (SGF, pH 1) and simulated colonic fluid (SCF, phosphate buffer at pH 7.4) at 37 ± 1 °C. At specific time intervals, the immersed microcapsules were removed from the swelling solutions. They were blotted on filter paper to absorb extra water on the surface and weighed immediately. The swelling ratios of the microcapsules were calculated using the eqn (2):where:

W_s = weight of the swollen magnetic microcapsules at the time t;

W_d = initial weight of the magnetic microcapsules.

2.9. In vitro drug release studies

In vitro drug release studies were carried out at two different pH solutions (SGF and SCF). For each experiment, 50 mg of dried microcapsules were placed in the aqueous buffer solutions (5 mL). The mixture was introduced into a cellophane membrane dialysis bag. The bag was closed and transferred into a beaker containing 25 mL of the same aqueous buffer solution maintained at 37 ± 1 °C. The external solution was continuously stirred. At appropriate time intervals, 3 mL of the solution was withdrawn and analyzed spectrophotometrically at the absorption maximum (λ_max) of the drug. The amounts of released drug were determined by using a standard calibration curve obtained under the same conditions. The withdrawn solutions were replaced with the same volume of fresh buffer solutions to keep the total volumes constant. The in vitro release profiles were established in both SGF (pH 1) and SCF (pH 7.4).

3. Results and discussion

3.1. Preparation of magnetic microcapsules

The formulations, preparation steps and drug encapsulation efficiencies of magnetic microcapsules are summarized in Table 1 and schematically shown in Scheme 2. At step 1, drug and MNPs were encapsulated into the Alg network by formation of Alg–Ca²⁺ complex (preparation of M.Alg). The M.Alg/Cs microcapsules were prepared by using LbL method that led to the formation of PECs between Alg and Cs. In PECs, physical interactions are the most important interaction which in this case were electrostatic adsorption forces between the anionic groups of Alg (–COO⁻) and cationic groups of CS (–NH₃⁺). Using this method (LbL), the anionic groups of CMC interacted with cationic groups of Cs and then CMC layer coated the M.Alg/Cs microcapsules and M.Alg/Cs/CMC was prepared (Scheme 2). As it can be seen, all procedures were carried out under mild, green and environmentally friendly conditions without using any toxic or hazardous materials.

Table 1 Microcapsules formulations and encapsulation ratios of prepared microcapsules

Microcapsules formulations	Step 1	Step 2	Step 3	Encapsulation efficiency (%)
M.Alg	CaCl2 solution	—	—	92.4 ± 1.7
M.Alg/Cs	CaCl2 solution	Cs solution	—	83.7 ± 1.8
M.Alg/Cs/CMC	CaCl2 solution	Cs solution	CMC solution	75.1 ± 1.2

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Scheme 2 Preparation of layered microcapsules and the interaction between oppositely charged polymers.

The wet and dried forms of microcapsules containing MNPS and without MNPs are shown in Fig. 1a and b. If too much amount of drug is used, the extra amount will leach out from the microcapsules during drying processes and drug crystals will appear on the surface of the microcapsules (Fig. 1c). Moreover, if hardening process (i.e. immersing in calcium chloride solution, step 1) is carried out in the presence of a permanent magnet, MNPs encapsulated into the microcapsules, will gather at the one side next to the permanent magnet (Fig. 1d) but the permanent magnet could be used after complete hardening of microcapsules. Fig. 1e shows the tendency of magnetic microcapsules towards the external magnet.

Fig. 1 Digital images of (a) wet microcapsules; (b) dried microcapsules; (c) drug crystals on microcapsules; (d) MNPs gathered on one side of microcapsules; (e) microcapsules are attracted toward the external magnet.

3.2. FT-IR characterization

The FT-IR spectra are shown in Fig. 2. The FT-IR spectrum of pure chitosan shows the strong peak at 3441 cm⁻¹ (O–H stretching), the weak band at 2853–2923 cm⁻¹ (C–H stretching) and the absorption peak at 1637 cm⁻¹ (carbonyl stretching). The peaks at 1458 and 1261 cm⁻¹ belong to the N–H stretching of the amide and ether bonds respectively. The bridge oxygen bands were observed at about 1093 and 850 cm⁻¹.^54–56 In the spectrum of CMC, the bands at 2903, 1626 cm⁻¹ and 1058 cm⁻¹ are related to the stretching vibration of CH₂, carboxyl and primary alcoholic (CH₂OH) groups, respectively. The bands in the region 1327–1410 cm⁻¹ are related to symmetrical deformations of CH₂ and COH groups. The weak bands at around 715 cm⁻¹ come from ring stretching and ring deformation of α-D-(1-4) and α-D-(1-6) linkages.^55,57

Fig. 2 The FT-IR spectra of (a) Cs; (b) CMC; (c) M.Alg; (d) M.Alg/Cs; (e) M.Alg/Cs/CMC.

The spectrum of M.Alg shows the bands at 3423 cm⁻¹ and 2924 cm⁻¹ for O–H and C–H stretching vibration, respectively. The strong bands at 1633 and 1431 cm⁻¹ correspond to asymmetric and symmetric –COO⁻ stretching vibration of the carboxylate groups. The peaks at 1085 cm⁻¹ and 1030 cm⁻¹ are assigned to stretching vibration of C–O–C bridge in uronic acids.^58–60 The spectra of layered microcapsules (PECs) are similar to each other and no big differences can be seen (Fig. 2c–e). This is expected because the “Coulomb forces” are the most and primary bonding force between the polymers in PEC microcapsules and also confirms that no new covalent bonds have been formed during the preparation of these PEC microcapsules. Though the spectra of microcapsules are similar and most of the peaks overleaped, there are still some differences in Fig. 2c, in comparison with Fig. 2d and e that confirms that PECs (layered microcapsules) are formed. First, in the spectrum of M.Alg, the original sharp hydroxyl peak (3423 cm⁻¹, Fig. 2c) became broader and moved to the lower frequencies (3403 and 3395 cm⁻¹, Fig. 2d and e), that could be explained by the superposition of the stretching vibration of υ –NH₂ groups in Cs. Second, the peaks at 1633 and 1431 cm⁻¹ (in Fig. 2c) which correspond to asymmetric and symmetric –COO⁻ stretching vibration of the carboxylate groups in Alg, moved to 1614 and 1407 cm⁻¹ (in the Fig. 2d) and to 1618 and 1410 cm⁻¹ (in the Fig. 2e). It was caused by the interactions between carboxylate groups with ammonium groups.⁶¹ The peaks appeared in 1730 and 1728 cm⁻¹ in the spectrum of M.Alg/Cs and M.Alg/Cs/CMC, which does not exist in the spectrum of M.Alg, are originated from Cs (marked by red arrows in the Fig. 2a, d and e). This confirms the existence of Cs layers in these layered microcapsules. Finally, in the spectrum of M.Alg/Cs/CMC (Fig. 2e), the peak at 1339 cm⁻¹ (green arrows in the spectra b, e) which is not be found in the spectrum of M.Alg/Cs (Fig. 2d), comes from CMC and shows that CMC layer has been successfully coated on the surface of M.Alg/Cs microcapsules.

3.3. Differential scanning calorimetry (DSC)

Differential scanning calorimetry (DSC) is usually employed to determine the thermal properties and possible interactions between the components. The DSC thermograms of Cs, Alg, CMC and prepared magnetic microcapsules are shown in Fig. 3. The broad endothermic peaks at 85–105 °C in the all thermograms are related to the water evaporation. The thermogram of pure Alg shows a wide exothermic peak at 250 °C that corresponds to the decomposition of Alg.⁶² The decomposition peak of Alg in M.Alg microcapsules shifts to the higher temperature (305 °C) owing to cross-linking process in presence of Ca²⁺. In the thermogram of pure Cs, the exothermic peak at about 310 °C can be attributed to its decomposition.⁶³ The DSC curve of CMC shows a broad exothermic peak at 278 °C due to the decomposition of polymeric main chains.⁶⁴ The endothermic peaks at about 160 °C in the thermogram of drug loaded M.Alg, M.Alg/Cs and M.Alg/Cs/CMC microcapsules, are related to naproxen which definitely corresponds to its melting point.⁶⁵ The presence of this peak in these thermograms confirms the existence of naproxen crystals in the microcapsules. The exothermic decomposition peaks of pure Alg, Cs and CMC are not exactly observed in the thermograms of microcapsules (i.e. M.Alg, M.Alg/Cs and M.Alg/Cs/CMC). Instead, exothermic events with different shapes and positions have appeared. These differences between the thermograms of pure polymers and microcapsules, represent the presence of ionic interactions, which could lead to the formation of new structures (PECs) with different thermal characteristics.⁴⁶

Fig. 3 The DSC curves of (a) Alg; (b) Cs; (c) CMC; (d) M.Alg; (e) M.Alg/Cs; (f) M.Alg/Cs/CMC.

3.4. Morphologies

Generally, the wet (or swollen) state of prepared microcapsules were spherical with a diameter of about 2 mm and possessed smooth surfaces. After air drying process, the size of microcapsules was decreased, but their spherical shapes were kept. It can be observed in micrographs of wet and dried microcapsules recorded by optical microscope (Fig. 4a and b).

Fig. 4 Images recorded by optical microscope (a) wet magnetic microcapsules; (b) dried microcapsules.

The SEM images of magnetic microcapsules (M.Alg, M.Alg/Cs and M.Alg/Cs/CMC) are shown in Fig. 5. Although the drying process had been performed under mild conditions (at room temperature), the loss of water changed the ideal spherical form of all microcapsules and also some cracks and wrinkles were formed. These deformations can be attributed to the drying process. Smrdel et al.⁶⁶ studied the influence of drying methods on the morphology of microcapsules. These shape deformations are unavoidable because all these microcapsules are hydrogel (with high water content) and water loss during the drying process leads to contraction and shrinkage of the hydrogel network. The rough outer surface with a sandy appearance of all microcapsules may be explained by the existence of drug crystals. This is expected because the drug had been suspended in Alg solution before encapsulation.⁶⁷

Fig. 5 The SEM images of (a₁–a₃) M.Alg microcapsules; (b₁ and b₂) M.Alg/Cs microcapsules; (c₁–c₃) M.Alg/Cs/CMC microcapsules; (d₁) non-modified microcapsules without MNPs and (d₂) MNPs into the M.Alg/Cs microcapsules.

In the SEM of M.Alg microcapsules, too many wrinkles, fractures and cracks can be observed (as it is shown in Fig. 5a₁–a₃). However, after coating with Cs, M.Alg/Cs microcapsule shows a surface modification (Fig. 5b₁ and b₂). The Alg/Cs microcapsule shows a heterogeneous surface with less wrinkles and cracks.^67,68 In comparison with M.Alg and M.Alg/Cs, roughness seems to be relatively lower in M.Alg/Cs/CMC (Fig. 5c₁–c₃). Coating with CMC turns microcapsules into being smoother and having fewer cracks (Fig. 5a₃ and c₃). This change might be attributed to the formation of CMC layers on the rough surface of microcapsules.⁶⁹ The close SEM image of M.Alg/Cs (Fig. 5d₂) reveals the surface smoothness and MNPs in comparison with the bare microcapsule (Fig. 5d₁).

3.5. XRD

The crystalline structure of M.Alg/Cs/CMC microcapsules was characterized as depicted in Fig. 6. The indices (440), (511), (422), (400), (311), (220) revealed in this figure, can be well indexed to the inverse cubic spinel structure of MNPs (Fe₃O₄). This confirms that the encapsulation of MNPs did not change their crystal phase.⁶¹

Fig. 6 The XRD spectrum of M.Alg/Cs/CMC microcapsules.

3.6. Swelling studies

Swelling is one of the most important properties of polymeric microcapsules (especially alginate microcapsules) that is usually associated with the drug release behavior. Swelling degree is the trend and ability of hydrogels to absorb water. As a result of that an increase in the size and weight of microcapsules happens. To study this in PEC microcapsules, some points should be noticed (e.g. the nature of ionic groups, solubility of polymers, the way ionic groups interact with each other, porosity, etc.). The swelling studies were carried out in two different media (SGF, SCF) and results show that the pH of media dramatically affected on water absorbing. It can be seen in Fig. 7 that, the swelling ratios of prepared microcapsules at higher pH are more than that of lower pH. In acidic media (SGF), all investigated magnetic microcapsules keep their structures. It can be explained by the maintenance of Alg–Ca²⁺ complexes in microcapsules core, that do not let microcapsules disintegrate.

Fig. 7 The swelling ratios of magnetic microcapsules in (a) SGF and (b) SCF.

The M.Alg/Cs microcapsules are formed by polyelectrolyte complex (PEC), in which the carboxylate groups of Alg electrostatically interact with the ammonium groups of Cs. As it is shown in Fig. 7a, in comparison with M.Alg, the M.Alg/Cs has higher swelling ratios although formation of this PEC reduced the porosity and fractures in the M.Alg/Cs microcapsules (mentioned in SEM section). To clarify this, the interactions between the polymers (Alg and Cs) should be noticed. Firstly, in the acidic medium, the –NH₂ groups of Cs are protonated. The proportion of –NH₂ to –NH₃⁺ in SGF for Cs is about 10^−5.5 (|NH₂|/|NH₃⁺| = 10^−5.5). This amount can be calculated via Henderson–Hasselbalch equation (eqn (3)). Where for Cs, the pH and pK_a are 1 and 6.4 respectively.

pH = pK_a + log(|A⁻|/|HA|) (3)

Owing to the existence of these cationic groups (–NH₃⁺), a repulsive force in Cs chains could be revealed. Secondly, the protonation of –NH₂ and –COO⁻ groups lead to weakening of ionic interactions between Alg and Cs. Thirdly, in acidic media, Cs becomes more hydrophilic and then it is inclined to absorb water. All these events lead to an increase in water penetration in M.Alg/Cs that result in the high swelling degree.

In M.Alg/Cs/CMC, the decreases in swelling ratios in comparison with M.Alg/Cs can be explained by both decreasing of porosity and protonation of the –COO⁻ groups on the outer CMC layer (|COO⁻|/|COOH| = 10^−3.3, pH = 1, pK_a = 4.3). The protonation of –COO⁻ groups makes CMC layer be more hydrophobic, hence the water diffusion into microcapsules is decreased. In addition, all prepared microcapsules (i.e. M.Alg, M.Alg/Cs and M.Alg/Cs/CMC) are stable and do not erode in SGF. This can be explained by the existence of Alg–Ca²⁺ complex that remains intact in SGF.

In SCF, all prepared microcapsules swell promptly (Fig. 7b). The quick water uptake followed by entire disintegration of the M.Alg microcapsules takes place and it can be explained by the both chelating role of phosphate ions and electrostatic repulsion force between –COO⁻ groups of Alg at the higher pH.⁷⁰ The affinity of phosphate ion for calcium is higher than that of Alg. It causes breaking the Alg–Ca²⁺ complex followed by complete disintegration and dissolution of the M.Alg microcapsules.

The solution penetration into the M.Alg/Cs microcapsules are restricted in SCF. This is caused by two reasons: first, porosity is decreased; and second, the –NH₃⁺ groups of Cs are deporotonated and therefore become more hydrophobic layer (|NH₂|/|NH₃⁺| = 10¹, in SGF).

The swelling ratios are not efficiently reduced in M.Alg/Cs/CMC. The carboxylate groups of CMC are deprotonated (|COO⁻|/|COOH| = 10^3.1, in SGF) and as a result, the CMC layer become more hydrophilic layer and solution diffusion into the microcapsules would not be considerably reduced.

Covering Alg cores with Cs and CMC not only noticeably reduce the swelling ratios, but also these additional layers prevent M.Alg/Cs and M.Alg/Cs/CMC from complete erosion. These modifications certainly show the importance of these PECs layers.

3.7. In vitro studies

Drug encapsulation efficiencies of the all magnetic microcapsules are shown in Table 1. These high values of encapsulation efficiencies, which are similar to those of other works,⁴⁶ can be explained by the; firstly, quick ionotropic gelation process that happens instantly after dropping Alg into hardening solution (step 1); and secondly, because of the low solubility of naproxen in the acidic solution of Cs (step 2).⁷¹ However, drug encapsulation value of M.Alg shows that complete drug encapsulation is not happened even during step 1. The lower amount of drug encapsulation efficiencies for M.Alg/Cs and M.Alg/Cs/CMC (in comparison with M.Alg), can be attributed to the diffusion of drug from microcapsules into the Cs and CMC solutions during the LbL process (steps 2 and 3).

Comparing release percentages in both SGF and SCF (Fig. 8) reveals that the release percentages in SGF were by far lower than that of SCF. It could be explained by following reasons: (a) all microcapsules have the lower swelling degrees at SGF; (b) the Alg part, that encapsulates the drug, has a stable structure and would not be eroded in SGF; (c) the solubility of naproxen in acidic media is low.

Fig. 8 The drug release profiles magnetic microcapsules as a function of time at 37 ± 1 °C; (a) in SGF and (b) in SCF.

In SGF, the release percentages of M.Alg are lower than M.Alg/Cs. As mentioned in swelling studies section, Cs layer become more hydrophilic; the interaction between Alg and Cs would be decreased; and the repulsion forces between Cs chains take place, therefore Cs could not effectively halt or reduced solution diffusion and the solution could pass easily through the Cs layer. To modify these, the CMC layer was applied and it somewhat reduced the release percentages of microcapsules in SGF. In acidic media, –COO⁻ groups of CMC are protonated and become hydrophobic, subsequently M.Alg/Cs/CMC could obstacle water diffusion more effectively than other microcapsules.

In SCF, the M.Alg microcapsules released most of its encapsulated drugs in 180 min (about 85%). As mentioned before, this can be explained by disintegration and erosion of Alg–Ca²⁺ complexes due to chelating effect of phosphate ions. In comparison to M.Alg, the release percentages of M.Alg/Cs microcapsules are obviously lower. In alkaline media, Cs become an insoluble and more hydrophobic polymer (owing to deporotonation of –NH₃⁺ groups) thus solution diffusion and drug release ratios are decreased. In addition, the incorporation of a Cs layer can hamper the drug diffusion by steric hindrance. Drug release percentages for M.Alg/Cs/CMC are a little lower than that of M.Alg/Cs. The CMC layer in alkaline pH is more hydrophilic and solution can easily pass through this layer, therefore there are no big differences between M.Alg/Cs/CMC and M.Alg/Cs. Compared to the M.Alg/Cs, M.Alg/Cs/CMC has lower release ratios, this might be attributed to the fact that M.Alg/Cs/CMC covered with the extra layer (CMC) compared to M.Alg/Cs, as a result drug must traverse longer distance to reach the surface.

Briefly, comparing of the release percentages shows that formation of these polymeric layers played an important role on the pH-sensitivity of microcapsules with alginate base and reduced the release ratios.

4. Conclusions

In this study magnetic pH-sensitive microcapsules were prepared as a drug carrier under entirely green and environmentally friendly conditions. Not only safe and natural biopolymers were used, but also a completely economical, green, safe and organic solvent free procedure was employed. These layered microcapsules were prepared by LbL method. In this method, PECs are formed between oppositely charged biopolymers (i.e. Alg and CMC as a negatively charged polymer and in contrary, Cs as a positively charged polymer). Using this method, Cs and CMC layers were successfully coated on the microcapsules with Alg base and as a result, the porosity of microcapsules were reduced. The microcapsules had high encapsulation efficiencies because of the fast drug encapsulation and low solubility of naproxen. The results show that these coated microcapsules are completely pH-sensitive and potentially can be used as stimuli responsive carriers for colon specific drug delivery. The M.Alg/Cs/CMC microcapsules had lowest release percentages in both SGF and SCF.

Acknowledgements

The office of the research vice chancellor of Azarbaijan Shahid Madani University has supported this work.

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