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kappa-carrageenan hydrogel beads for controlled protein release: Effect of pH and crosslinking agent
Journal of Drug Delivery Science and Technology 56 (2020) 101551
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pH-responsive double network alginate/kappa-carrageenan hydrogel beads for controlled protein release: Effect of pH and crosslinking agent
T
Selin Sarıyer1, Dilek Duranoğlu∗, Özlem Doğan, İlknur Küçük Yildiz Technical University, Chemical Engineering Department, 34220, Esenler, İstanbul, Turkey
ARTICLE INFO
ABSTRACT
Keywords: Alginate Kappa-carrageenan pH-responsive hydrogel Double network hydrogel Encapsulation BSA release
Bovine Serum Albumin (BSA) was encapsulated into pH-responsive alginate/kappa(κ)-carrageenan double network hydrogel beads at different pHs and CaCl2/KCl ratios. Controlled release of BSA was performed in the simulated gastric fluid (SGF) and then in simulated intestinal fluid (SIF). Alginate reinforced the mechanical properties while double network with kappa-carrageenan made slow down the release of BSA into SIF. Sulfate groups on kappa-carrageenan improved the resistivity against phosphate buffer solution. Encapsulation and BSA release was manipulated by varying pH and crosslinking concentration. The highest encapsulation efficiency (83–89%) was obtained with the hydrogel beads prepared at the lower pH than isoelectric point of BSA, then 67–72% of them released in SIF in 4–5 h while lower than 10% of BSA released in SGF, hence, protein delivery targeted intestinal was achieved. BSA release exhibited a coupling of diffusion and polymer relaxation mechanism without burst release.
1. Introduction Therapeutic proteins have significant role in the treatment of many diseases, for example, human insulin is beneficial for diabetes, and nivolumab and nembrolizumab can be used for cancer immunotherapy [1,2]. However, there are many difficulties on the application of protein drugs like denaturation, aggregation, and hydrolyzation [3]. Therefore, efficacy, stability and biological activity of proteins can reduce when they are exposed to different environmental conditions such as humidity, temperature, pH, light, oxygen, gastrointestinal fluids. In order to overcome these problems, protein drugs can be encapsulated in a three dimensional, water or physiological fluid-swollen chemically or physically crosslinked macromolecular network, which is known as hydrogel. Synthetic and natural polymers are widely used forproducing hydrogels. Polysaccharide such as chitosan, alginate, carrageenan, hyaluronic acid, cellulose based hydrogels are gaining interest because of their great biodegradability and biocompatibility. Furthermore, there is more interest on drug delivery studies and effective drug delivery systems using polysaccharides [4,5]. Alginate is a natural anionic polysaccharide and biopolymer extracted from brown algae, and is comprised of varying quantity of 1,4′-linked β- D -mannuronic acid (M) and α-l-guluronic acid (G) monomers [6]. Alginate hydrogel is formed by physically crosslinking with divalent cations such as calcium ion, which is the most commonly
used because of its biocompatibility [7]. Alginate based hydrogels are extensively used as wound dressings in biomedical application [8], cell transplantation in tissue engineering and delivery of protein/drug in controlled release systems [9]. Another significant properties of alginate is pH sensitivity. pH dependence of alginate beads makes them suitable for designing oral delivery system. In gastric environment, alginate based hydrogel shrinks and cannot allow the entrapped bioactive proteins to release, but in intestinal environment, alginate beads become swollen so that encapsulated protein or drugs can be released [10]. Kappa carrageenan (κ-car) is a natural anionic polysaccharide, and linear sulphated biopolymer obtained from the tropical seaweed. It is composed of alternating 3-linked β-d-galactopyranose (G-units) and 4-linked α-d-galactopyranose (D-units) or 4-linked 3,6-anhydro-α-dgalactopyranose (DA-units) [11]. In biomedical applications, appropriate forms of the carrageenan based system such as beads, microcapsules and microspheres have been widely used to release of drugs or proteins because of its biocompability, biodegradability, the ability to form gel and thermo reversibility [12]. Gelation of κ-car takes place in two steps: in the first step, helix structure is formed at 80–90 C, and sulfate group of κ-car is neutralized by cations such as Ca+2, Na+ and K+, in the second step, three dimensional network is formed by decreasing the temperature to 50 °C [13]. It has been reported that stronger κ-car gels were obtained in the presence of KCl
Corresponding author. E-mail addresses: [email protected], [email protected] (D. Duranoğlu). 1 Present address: Gebze Technical University, Chemical Engineering Department, 41400, Gebze, Kocaeli, Turkey. ∗
https://doi.org/10.1016/j.jddst.2020.101551 Received 5 December 2019; Received in revised form 17 January 2020; Accepted 27 January 2020 Available online 30 January 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
Journal of Drug Delivery Science and Technology 56 (2020) 101551
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compared to other salts such as NaCl, MgCl2, LiCl, SrCl2 and CaCl2 [14]. In particular, potassium ions promote the formation of an ionic bond with the sulfate group from one galactose residue and electrostatic interaction with an anhydrous-O-3,6-ring of another galactose residue, whereas divalent calcium ions are crosslinked with sulfate groups on neighboring single or double helices. These ion-mediated forces and intermolecular associations enhance chain stability [15,16]. Furthermore, gel–sol and sol–gel transition temperature is higher in the presence of potassium ions due to the lower electronegativity comparing to other monovalent ions (K+:0.82; Na+:0.93; Li+:0.98) [14]. The main purpose of this study is to synthesize biocompatible, mechanically strength, pH-resposive double crosslinked hydrogel networks in order to protect protein from acid hydrolyze and proteolytic breakdown passing through the gastrointestinal tract. Herein, polymer double network with a synergistic effects of alginate and κ-carrageenan was synthesized with a simple method. The effect of pH and the ratio of crosslinking agents (K+ and Ca2+ ions) on encapsulation efficiency of BSA, and on release behavior in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was investigated comprehensively. Up to now, several studies have been published about the entrapment of betamethasone acetate [17], riboflavin [18], and α-amylase [19] in physically crosslinked alginate-κ-carrageenan hydrogel network. To the best of our knowledge, the effect of pH on encapsulation efficiency and BSA release profile of alginate/ĸ-carrageenan hydrogel bead has not been reported in the literature.
Loading capacity (mg/g) =
2.3. BSA release studies and release kinetics BSA release studies were performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively. 2 g of sodium chloride (NaCl) was dissolved in 500 mL of distilled water, then 7 mL of hydrochloric acid (37.4%) was added and the final solution was completed to 1 L with distilled water in order to prepare SGF (pH = 1.2) [16]. 6.8 g of potassium dihydrogen phosphate (KH2PO4) was dissolved in 500 mL of distilled water, then 190 mL of 0.2 M sodium hydroxide was added, and the final solution was completed to 1 L with distilled water in order to prepare SIF (pH = 7.4) [17]. BSA release experiments were carried with 5 g of BSA loaded wet beads placed in Erlenmeyer flask containing 100 mL of SGF. After 2 h stirring at 150 rpm at 37 °C, the beads were taken and filtered from SGF and transferred to 100 mL of SIF. 1mL sample was withdrawn from release medium at specified time intervals and filtered through a 0.45 μm membrane filter. BSA concentration was measured in release medium using UV spectrophotometry at 280 nm. Cumulative BSA release was calculated using the following Equation (3):
2.1. Materials
Cumulative Release (%) =
Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. All other reagents have analytical grade and were purchased from Sigma-Aldrich. Sodium alginate (NaAlg) and Kappa-carrageenan (κ-car) were provided by Will Powder and Molecular Gastronomy Recipes, respectively.
Mt = kp × t n M
Mf
×100
(3)
(4)
where, n: diffusional exponent, which defines the mechanism of drug release; kp: rate constant; t: time.
NaAlg and κ-car aqueous dispersions were prepared separately. NaAlg powders (2% w/v) were dissolved in 70 mL of distilled water under constant stirring overnight. κ-car powders (2% w/v) were dissolved in 30 mL of distilled water, at 80 °C for an hour, then the temperature was reduced to 25 °C under constant stirring overnight. After 24 h, NaAlg and κ-car solutions were mixed together for 2 h with continuous stirring to obtain a uniform solution. 5 mL of 1% BSA solution was added to the NaAlg- κ-car mixture and pH of the mixture was adjusted to desired pH value by adding diluted HCl solution slowly. BSA loaded beads were formed by dropping the mixture into 250 mL of salt solution (%CaCl2:%KCl) using a peristaltic pump (Masterflex L/S; 5 mL/min flow rate; 16 cm tube diameter). The beads were kept in salt solution for 10 min for gelation and then diluted by 250 mL of distilled water. BSA loaded beads were filtrated from the salt solution and then kept in distilled water for 48 h for further usage. Schematic illustration of preperation of BSA loaded double network Alg-κ-car hydrogel beads can be seen in Fig. 1. In order to determine encapsulated BSA, BSA concentration was analyzed by using UV spectrophotometry (Analytic Jena Specord 200) at 280 nm. BSA encapsulation efficiency was calculated using the following Equation (1):
Mi
Mt ×100 M
where, Mt: the amount of BSA (mg) released after time t; M∞: the amount of BSA (mg) loaded in the beads. BSA release kinetic was analyzed by Ritger-Peppas Model [20]:
2.2. Preparation of BSA loaded Alg-κ-car beads
Mi
(2)
The FTIR–ATR spectra of the samples were obtained using a Bruker Optics Alpha-P, in the 400–4000 cm−1 region and at a resolution of 4 cm−1. Spectra were obtained by collecting the average of 20 scans. Surface morphology of the beads was observed by using Scanning Electron Microscope (SEM, Zeiss EVO LS 10). The beads were coated with gold and operated at an accelerating voltage of 5 kV.
2. Experimental
Encapsulation Efficiency (%) =
amount of encapsulated BSA (mg ) total amount of wet beads (g )
3. Results and discussion BSA loaded beads prepared with various CaCl2:KCl concentration, and pH are shown in Table 1 with the corresponding encapsulation efficiencies and loading capacities. 3.1. Characterization of hydrogel beads Surface morphology of hydrogel beads was observed by using SEM (Fig. 2). As can be seen that Alg-κ-car hydrogel beads (prepared with the 2% of CaCl2) have no large pores and present tight surface (Fig. 2a) although BSA loaded hydrogel bead (B7) has a rough surface (Fig. 2b). It was observed that BSA bound to bead structure and was trapped in the bead, however, accumulation of BSA on the surface was not observed clearly (Fig. 2b). Functional groups of BSA, hydrogel beads and BSA loaded hydrogel beads (B7) were determined by using FT-IR spectrometer (Fig. 3). FTIR-ATR spectrum of BSA loaded hydrogel bead (B7) almost overlapped with the spectrum of hydrogel bead. The characteristic peaks of BSA at 1639 and 1521 cm−1 which are due to C]O stretching vibrations of the amide I (-NH2) group and bond between N–H of amide II (-NH-) group [21] was not observed on the FTIR-ATR spectrum of BSA loaded hydrogel (Fig. 3).
(1)
where Mi: the amount of BSA (mg) added to NaAlg- κ-car mixture; Mf: the amount of BSA (mg) in salt solution. BSA loading capacity of hydrogels was calculated by using Equation (2): 2
Journal of Drug Delivery Science and Technology 56 (2020) 101551
S. Sarıyer, et al.
Fig. 1. Schematic illustration of preperation of BSA loaded Alg-κ-car hydrogel beads.
groups on the alginate molecules are available for cross-linking with calcium ions [26]. Higher amount of anionic groups on NaAlg and -κ-car at pH 7.5 could be resulted with the highest cross-linking, which can affect the BSA encapsulation of hydrogel beads in a positive way. It can be concluded that electrostatic attraction and higher crosslinking degree are dominant for BSA encapsulation on hydrogel beads prepared at pH below and above isoelectric point, respectively.
Table 1 BSA loaded beads. Formulation
B1 B2 B3 B4 B5 B6 B7 B8 B9
pH
4.2 4.2 4.2 4.8 4.8 4.8 7.5 7.5 7.5
Salt Concentration
Encapsulation Efficiency (EE)
BSA Loading Capacity
CaCl2:KCl (%:%)
(%)
(mg BSA/g bead)
2:0 2:2 4:4 2:0 2:2 4:4 2:0 2:2 4:4
83 85 89 29 36 53 62 64 68
18.4 18.4 18.6 5.5 7.1 10.9 11.7 12.2 15.4
3.3. Effect of crosslinking salt concentration on BSA encapsulation BSA encapsulation efficiency of hydrogel beads prepared at different cosslinking salt concentration ratios (CaCl2:KCl) was also investigated. Fig. 4 shows that the BSA encapsulation efficiency increases as the salt concentration ratio increases at each pH value (pH 4.2, 4.8 and 7.5). The encapsulation efficiency of BSA loaded beads prepared at pH 4.2 and pH 7.5 showed a slight increase with an increase in the salt concentration. Low encapsulation efficiency at low salt concentrations can be explained with the insufficient cross-linking. During bead formation, BSA can be diffused out of insufficient cross-linking beads. It has been reported that the BSA encapsulation efficiency of alginate-chitosan beads loaded with BSA increased by increasing CaCl2 concentration [27]. The increase in the encapsulation efficiency with the salt concentration of BSA loaded beads prepared at isoelectric point (pH 4.8) is much higher than that of the others. This can be explained that, due to the weak electrostatic attraction between polymer and BSA, with the increasing salt concentration, cross-linking becomes more effective on encapsulation efficiency at isoelectric point.
3.2. Effect of pH on BSA encapsulation BSA can electrostatically interact with the pH-sensitive alginate and κ-car biopolymers. pKa value of mannuronic and gluronic units of alginate are 3.38 and 3.65, respectively [18], and the pKa value of κ-car is about 2 [18,22]. Thus, electrostatic interaction between BSA and alginate-κ-car can be manipulated by varying the pH. The effect of pH, below (pH 4.2), above (pH 7.5) and at isoelectric point (pH 4.8), on encapsulation efficiency was investigated. BSA molecules have net zero charge at isoelectric point (pH 4.8) while positively charged at pH 4.2, and negatively charged at pH 7.5 [23]. BSA encapsulation efficiencies obtained at different pHs are shown in Fig. 4. As can be seen that the lowest encapsulation efficiency was obtained at pH 4.8 while the highest encapsulation efficiency was obtained at pH 4.2. This can be interpereted with the interactions of charges of both hydrogel beads and BSA. In the values below the isoelectric point of BSA, BSA molecules are positively charged while κ-car and NaAlg are negatively charged [24]. Therefore, encapsulation efficiency reached at the highest value (89%) due to the electrostatic attraction between negatively charged alginate and κ-car in the beads and positively charged BSA molecules at pH 4.2. Similarly, it has been reported that BSA loading efficiency was higher at lower pH values than isoelectric point of protein because of the electrostatic interaction between BSA and alginate [25]. At pH 7.5, encapsulation efficiency was obtained lower (68%) than that of pH 4.2 since the electrostatic repulsion is dominant between negatively charged BSA and ionized carboxylic groups in the hydrogel beads. However, encapsulation efficiency obtained at isoelectric point of BSA is the lowest (53%) due to the decreasing electrostatic attraction between ionized carboxylic and sulfonic groups in the beads and neutral BSA molecules. At the pH values above the isoelectric point of BSA, there must be more strong interaction than the electrostatic repulsion of negatively charged groups. As it is known that anionic
3.4. BSA release studies 3.4.1. BSA release in SGF BSA release diffusion from hydrogel beads is controlled by both the swelling behavior and mechanical strength of the hydrogel beads and the chemical structure of the protein. After BSA release performed in SGF for the first 2 h, hydrogel beads were transferred into SIF. pH sensitive hydrogel beads became smaller due to the protonation of carboxylic and sulfate groups of alginate-κ-car at pH 1.2, so that BSA diffusion from hydrogel beads was very low, then, cumulative BSA release in SGF was obtained less than 10% (Figs. 5–7). To our knowledge, except [28], there is no previous study reported lower than 10% of BSA release from alginate contained hydrogel beads into SGF (Table 4). We thought that the presence of sulfate groups in the κ-car was significantly effective on inhibition of BSA release in SGF. Sulfate groups in κ-car can be resulted in higher crosslinking, i.e., smaller pore size, so that inhibiting BSA release. Similarly, Xu et al. decreased the BSA release into SGF from about 80% to 3% by adding SO42− ions into hydrogel structure [29]. 3
Journal of Drug Delivery Science and Technology 56 (2020) 101551
S. Sarıyer, et al.
Fig. 2. SEM images of a) Alg-κ-car hydrogel beads b) BSA loaded hydrogel beads (B7).
3.4.3. Effect of synthesis pH on BSA release As can be seen from Fig. 5, BSA release reached 72%, 80%, 21% from B1, B4, B7 hydrogel beads, 61%; 84%; 27% from B2; B5; B8 hydrogel beads, and 67%, 70%, 22% from B3, B6, B9 hydrogel beads, respectively. Among all formulations, highest BSA release percentage was obtained with the hydrogel beads prepared at isoelectric point, due to the weak electrostatic interactions between BSA molecules and hydrogel structure. It was followed by hydrogel beads prepared at slightly acidic pH (4.2). The least BSA release percentage was observed with the hydrogel beads due to the highest crosslinking ratio prepared at basic media (pH 7.5). However, the highest amount (in mg/g) of BSA release was obtained from the hydrogel beads, which were prepared with the highest encapsulation efficiency at pH 4.2 (Table 2). Salt concentration has little effect on BSA release from the beads prepared at pH 4.2 and 7.5, whereas, it is more effective on BSA release from the beads prepared at pH 4.8.
3.4.2. BSA release in SIF As can be seen BSA release profiles in Figs. 5–7 that, BSA was gradually released from the hydrogel matrix into SIF in 2–4 h. In SIF (pH 7.4), calcium ions were replaced by potassium and sodium ions. This change led to an increase in the swelling potential of the hydrogel beads, at the same time decreasing the stability of the hydrogel beads, hence, the amount of BSA released in SIF was greater. The porosity of the hydrogel matrix, the solubility of BSA, the swelling capacity of the hydrogel matrix and erosion in the releasing environment can affect BSA release in SIF [24]. Anal et al. stated that due to the chelating effect of phosphate ions, the disruption of the calcium alginate matrix was faster in the phosphate buffer solution at above pH 5.5 [27]. Li et al. obtained 180 μg BSA release in SIF (pH: 7.5) while 11 g BSA release in SGF (pH: 1.2) from 3 mg chitosan-carrageenan polyelectrolyte complex [30]. In another study, the BSA protein loaded on alginate-N,O carboxymethyl chitosan hydrogel beads was released at approximately 82–87% at pH 7.5 [31]. In the study of Sevgi and Aybige, the entire BSA encapsulated in the alginate-chitosan beads was released in the phosphate buffer solution (pH; 6.8) in 1 h [32]. 4
Journal of Drug Delivery Science and Technology 56 (2020) 101551
S. Sarıyer, et al.
Fig. 3. FTIR-ATR spectra of BSA, hydrogel beads and BSA loaded hydrogel.
Fig. 6. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = %2:%2. Fig. 4. Effect of pH and CaCl2:KCl (%:%) ratio on BSA encapsulation efficiencies.
Fig. 7. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = 4:4. Fig. 5. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = %2:%0.
According to the Ritger-Peppas model, each of the ln(Mt/M∞) versus ln(t) graphs produced linear curves. The correlation coefficients (0.913–0.995) indicated that chosen kinetic model succesfully fitted the experimental data (Table 3). The diffusional exponents (n) were obtained from the slope of the linear curves to verify the release mechanism of BSA from hydrogel beads. According to the Ritger and Peppas [20], for the swellable spherical matrices such as alginate-κ-car hydrogel beads, if the n value is equal or less than 0.43, there is a Case I (Fickian diffusion); if n is equal or greater than 0.85, case II (polymer
3.5. BSA release kinetics The release data of BSA from Alg-κ-car hydrogel beads into SIF medium were fitted to the Ritger-Peppas model and the release mechanism of BSA was investigated. Ritger-Peppas equation parameters and correlation coefficients were given in Table 3. 5
Journal of Drug Delivery Science and Technology 56 (2020) 101551
S. Sarıyer, et al.
in calcium and potassium crosslinked Alg-κ-car hydrogel beads at different pH and salt concentrations, was achieved by both the protein diffusion from beads and relaxation of hydrogel macromolecular structure. It was also observed during the period of BSA release in the SIF that the hydrogel beads first became swollen, very large and soft, and then lost their spherical structure by the time. Kaygusuz and Erim observed that 85% of BSA released from alginate hydrogel beads in 2 h [28], whereas, we observed the maximum 83% BSA release in 3 h (Fig. 6). It can be concluded that the burst release was inhibited, and the controlled release behavior of BSA was improved by using double network Alg-κ-car hydrogel beads in our study. This can be attributed to the highly crosslinked structure of prepared hydrogel as well as the resistivity against phosphate due to the sulfate groups on kappa-carrageenan.
Table 2 The results of BSA release study. Formulation
B1 B2 B3 B4 B5 B6 B7 B8 B9
Encapsulation Efficiency
Cumulative BSA release
Released BSA
BSA Release Yield*
(%)
(%)
(mg/g bead)
(%)
83 85 89 29 36 53 62 64 68
72 61 67 80 84 70 21 27 22
13.3 11.2 12.4 5.5 5.7 7.6 2.9 3.4 3.5
72 61 67 100 80 70 25 28 23
BSA Release Yield (%) =
the amount of released BSA (mg ) theinitial amount of BSA (mg )
4. Conclusion
× 100 .
pH-responsive double network alginate/kappa-carrageenan hydrogel beads were prepared by ion gelation method in order to encapsulate protein. BSA encapsulation was performed at three different pH values and different crosslinking salt solution concentrations. The highest BSA encapsulation efficiency (89%) was obtained with the B3 formulation, which was prepared with the highest cosslinking salt ratio (CaCl2:KCl = 4%:4%) at pH 4.2. The strong interaction between the positively charged BSA and negatively charged alginate and kappa carrageenan, and the high crosslinking salt concentration led to high encapsulation efficiency. BSA release from pH-responsive hydrogel beads in simulated gastric fluid (SGF) was negligible amount (less than 10%). In simulated intestine fluid (SIF), the highest BSA release occurred (13.3 mg/g) from the hydrogel beads prepared with the lowest crosslinking salt concentration (CaCl2:KCl = 2%:0%) at pH 4.2. According to Ritger-Peppas kinetic model, BSA release showed an anomalous release mechanism (diffusive behavior and polymer relaxation) i.e. BSA could release in intestine in a controlled manner. Preparing double network hydrogel beads with alginate and kappa-carrageenan as well as varying the synthesis pH and crosslinking salt concentration not only resulted with the high encapsulation efficiency and also controlled release of BSA in the intestine without burst release.
Table 3 Ritger-Peppas kinetic model parameters and correlation coefficients. Formulation
n
R2
B1 B2 B3 B4 B5 B6 B7 B8 B9 Alginate [28]
0.82 0.69 0.68 0.93 0.71 0.68 0.57 0.62 0.60 1.05
0.979 0.995 0.987 0.913 0.978 0.950 0.942 0.945 0.965 0.970
relaxation) transport mechanism dominates. In many cases, molecules release with a kinetic behavior that is dependent on the relative ratio of diffusion and relaxation mechanism, which is called as anomalous transport, where the diffusional exponent, n, is between the 0.43 < n˂0.85 [20]. According to obtained n values varying from 0.57 to 0.82 (Table 3), the release of the BSA from alginate-κ-car hydrogel beads exhibited both diffusion and polymer relaxation behavior. BSA diffusional exponents, n, obtained from the beads prepared at pH 4.2 and 4.8 decreased with the increasing salt concentration (n = 0.82 for B1; n = 0.68 for B3 and n = 0.93 for B4; n = 0.68 for B6). It can be explained that the stronger cross-linking by increasing the salt concentration inhibited the polymer relaxation, so diffusion was thought to be more effective than polymer relaxation for the high salt concentration. As a result, the release of BSA protein, which was loaded
CRediT authorship contribution statement Selin Sarıyer: Methodology, Investigation, Writing - original draft, Visualization. Dilek Duranoğlu: Supervision, Conceptualization, Methodology, Resources, Data curation, Writing - review & editing. Özlem Doğan: Conceptualization, Methodology, Data curation, Writing - review & editing, Funding acquisition. İlknur Küçük:
Table 4 BSA encapsulation and release in alginate based hydrogel beads. Hydrogel
Crosslinking agent
BSA Encapsulation Efficiency
BSA Release in SGF
BSA Release in SIF/PBS
Ref.
Alginate Alginate/P(CE-MAA-MEG) Alginate Alginate/N,O-carboxymethyl chitosan Alginate/Carboxymethyl Chitin Alginate Alginate/hydroxypropyl-methylcellulose Alginate/Poly(N-isopropyl acrylamide) Alginate/Chitosan
CaCl2 CaCl2 CaCl2 CaCl2 FeCl3 FeCl3 CaCl2·2H2O CaCl2 CaCl2 CaCl2/Na2SO4 CaCl2 CaCl2 BaCl2+ CaCl2 CaCl2 CaCl2 CaCl2/KCl
Not mentioned 72.3–78.6% 47–51% Not mentioned 95.4–96.8% 98.1% 69% 25% ≥97%
49.7% 13.4 Not experimented 25% 14–18% 15% Not experimented Not experimented ≥80% 1.76–2.35% 27% 9–13% Not experimented Not experimented Not experimented ≤10%
93.6% 60% 86-74% 80% 70% 80% 45% (pH = 7.1) 62% (pH = 7.4) 95% (pH = 6.8) 82.86–52.91% (pH = 6.8) 85% (pH = 7.2) 45–55% (pH = 7.2) 79.7% (pH = 7.4) 97.7% (pH = 7.4) 90% 67–72% 13.3–12.4 mg/g
[33]
Alginate Alginate/montmorillonite Alginate Alginate Alginate/Chitosan Alginate/Kappa-carrageenan
40% 78% 88% 86% Not mentioned 83–89% 18.4–18.6 mg/g
6
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Journal of Drug Delivery Science and Technology 56 (2020) 101551
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Conceptualization, Methodology, Investigation, Resources.
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Sung, Physically crosslinked alginate/N, O-carboxymethyl chitosan hydrogels with calcium for oral delivery of protein drugs, Biomaterials 26 (2005) 2105–2113, https://doi.org/10.1016/j. biomaterials.2004.06.011. [32] S. Takka, A. Gürel, Evaluation of chitosan/alginate beads using experimental design: formulation and in vitro characterization, AAPS PharmSciTech 11 (2010) 460–466, https://doi.org/10.1208/s12249-010-9406-z. [33] K. Wang, X. Xu, Y. Wang, G. Guo, M. Huang, F. Luo, X. Zhao, Y. Wei, Z. Qian, In vitro release behavior of bovine serum albumin from alginate/P (CE-MAA-MEG) composite hydrogel, Soft Mater. 8 (2010) 307–319, https://doi.org/10.1080/ 1539445X.2010.497076. [34] F. Yasmin, X. Chen, B.F. Eames, Effect of process parameters on the initial burst release of protein-loaded alginate nanospheres, J. Funct. Biomater. 10 (2019) 42, https://doi.org/10.3390/jfb10030042. [35] F.-L. Mi, H.-F. Liang, Y.-C. Wu, Y.-S. Lin, T.-F. Yang, H.-W. Sung, pH-sensitive behavior of two-component hydrogels composed of N, O-carboxymethyl chitosan and alginate, J. Biomater. Sci. Polym. Ed. 16 (2005) 1333–1345 https://doi:10.1163/ 156856205774472317. [36] X.W. Shi, Y.M. Du, L.P. Sun, J.H. Yang, X.H. Wang, X.L. Su, Ionically crosslinked alginate/carboxymethyl chitin beads for oral delivery of protein drugs, Macromol. Biosci. 5 (2005) 881–889 https://doi: 10.1002/mabi.200500063. [37] A. Nochos, D. Douroumis, N. Bouropoulos, In vitro release of bovine serum albumin from alginate/HPMC hydrogel beads, Carbohydr. Polym. 74 (2008) 451–457, https://doi.org/10.1016/j.carbpol.2008.03.020. [38] M.R. de Moura, F.A. Aouada, S.L. Favaro, E. Radovanovic, A.F. Rubira, E.C. Muniz, Release of BSA from porous matrices constituted of alginate–Ca2+ and PNIPAAminterpenetrated networks, Mater. Sci. Eng. C 29 (2009) 2319–2325, https://doi. org/10.1016/j.msec.2009.05.022. [39] M. Bajpai, P. Shukla, S. Bajpai, Ca (II)+ Ba (II) ions crosslinked alginate gels prepared by a novel diffusion through dialysis tube (DTDT) approach and preliminary BSA release study, Polym. Degrad. Stabil. 134 (2016) 22–29, https://doi.org/10. 1016/j.polymdegradstab.2016.09.027. [40] L. Xing, J. Sun, H. Tan, G. Yuan, J. Li, Y. Jia, D. Xiong, G. Chen, J. Lai, Z. Ling, Y. Chen, X. Niu, Covalently polysaccharide-based alginate/chitosan hydrogel embedded alginate microspheres for BSA encapsulation and soft tissue engineering, Int. J. Biol. Macromol. 127 (2019) 340–348, https://doi.org/10.1016/j.ijbiomac. 2019.01.065.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgement This work was supported by Yildiz Technical University Scientific Research Project Coordinator [project number 2013-07-01-KAP01]. References [1] M. Yu, J. Wu, J. Shi, O.C. Farokhzad, Nanotechnology for protein delivery: overview and perspectives, J. Contr. Release 240 (2016) 24–37 https://doi:10.1016/j. jconrel.2015.10.012. [2] P. Batista, P.M. Castro, A.R. Madureira, B. Sarmento, M. Pintado, Recent insights in the use of nanocarriers for the oral delivery of bioactive proteins and peptides, Peptides 101 (2018) 112–123 https://doi:10.1016/j.peptides.2018.01.002. [3] D.J. McClements, Encapsulation, protection, and delivery of bioactive proteins and peptides using nanoparticle and microparticle systems: a review, Adv. Colloid Interface Sci. (2018), https://doi: 10.1016/j.cis.2018.02.002. [4] W. Wei, J. Li, X. Qi, Y. Zhong, G. Zuo, X. Pan, T. Su, J. Zhang, W. Dong, Synthesis and characterization of a multi-sensitive polysaccharide hydrogel for drug delivery, Carbohydr. Polym. 177 (2017) 275–283, https://doi.org/10.1016/j.carbpol.2017. 08.133. [5] W.-F. Lai, Z.-D. He, Design and fabrication of hydrogel-based nanoparticulate systems for in vivo drug delivery, J. Contr. Release 243 (2016) 269–282, https://doi. org/10.1016/j.jconrel.2016.10.013. [6] W.R. Gombotz, S. Wee, Protein release from alginate matrices, Adv. Drug Deliv. Rev. 31 (1998) 267–285, https://doi.org/10.1016/S0169-409X(97)00124-5. [7] L. Agüero, D. Zaldivar-Silva, L. Pena, M.L. Dias, Alginate microparticles as oral colon drug delivery device: a review, Carbohydr. Polym. 168 (2017) 32–43, https:// doi.org/10.1016/j.carbpol.2017.03.033. [8] S.N. Pawar, K.J. Edgar, Alginate derivatization: a review of chemistry, properties and applications, Biomaterials 33 (2012) 3279–3305, https://doi.org/10.1016/j. biomaterials.2012.01.007. [9] K.Y. Lee, D.J. Mooney, Alginate: properties and biomedical applications, Prog. Polym. Sci. 37 (2012) 106–126 https://doi: 10.1016/j.progpolymsci.2011.06.003. [10] M. George, T.E. Abraham, Polyionic hydrocolloids for the intestinal delivery of protein drugs: alginate and chitosan—a review, J. Contr. Release 114 (2006) 1–14, https://doi.org/10.1016/j.jconrel.2006.04.017. [11] V.L. Campo, D.F. Kawano, D.B. da Silva Jr., I. Carvalho, Carrageenans: biological properties, chemical modifications and structural analysis–A review, Carbohydr. Polym. 77 (2009) 167–180, https://doi.org/10.1016/j.carbpol.2009.01.020. [12] L. Li, R. Ni, Y. Shao, S. Mao, Carrageenan and its applications in drug delivery, Carbohydr. Polym. 103 (2014) 1–11, https://doi.org/10.1016/j.carbpol.2013.12. 008. [13] J. Liu, X. Zhan, J. Wan, Y. Wang, C. Wang, Review for carrageenan-based pharmaceutical biomaterials: favourable physical features versus adverse biological effects, Carbohydr. Polym. 121 (2015) 27–36, https://doi.org/10.1016/j.carbpol. 2014.11.063. [14] S. Kara, E. Arda, B. Kavzak, Ö. Pekcan, Phase transitions of κ‐carrageenan gels in various types of salts, J. Appl. Polym. Sci. 102 (2006) 3008–3016, https://doi.org/ 10.1002/app.24662. [15] M. Nickerson, A. Paulson, Rheological properties of gellan, κ-carrageenan and alginate polysaccharides: effect of potassium and calcium ions on macrostructure assemblages, Carbohydr. Polym. 58 (2004) 15–24, https://doi.org/10.1016/j. carbpol.2004.06.016. [16] S. Distantina, Rochmadi, M. Fahrurrozi, Wiratni, Stabilization of kappa carrageenan film by crosslinking: comparison of glutaraldehyde and potassium sulphate as the crosslinker, International Proceedings of Chemical, Biological and Environmental Engineering (IPCBEE), 74 IACSIT Press, 2014, pp. 1–5 https://doi:10.7763/IPCBEE. 2014. [17] Z. Mohamadnia, M. Zohuriaan-Mehr, K. Kabiri, A. Jamshidi, H. Mobedi, pH-sensitive IPN hydrogel beads of carrageenan-alginate for controlled drug delivery, J. Bioact. Compat Polym. 22 (2007) 342–356, https://doi.org/10.1177/ 0883911507078519. [18] G.R. Mahdavinia, Z. Rahmani, S. Karami, A. Pourjavadi, Magnetic/pH-sensitive κcarrageenan/sodium alginate hydrogel nanocomposite beads: preparation, swelling behavior, and drug delivery, J. Biomater. Sci. Polym. Ed. 25 (2014) 1891–1906 https://doi: 10.1080/09205063.2014.956166.
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pH-responsive double network alginate/kappa-carrageenan hydrogel beads for controlled protein release: Effect of pH and crosslinking agent
T
Selin Sarıyer1, Dilek Duranoğlu∗, Özlem Doğan, İlknur Küçük Yildiz Technical University, Chemical Engineering Department, 34220, Esenler, İstanbul, Turkey
ARTICLE INFO
ABSTRACT
Keywords: Alginate Kappa-carrageenan pH-responsive hydrogel Double network hydrogel Encapsulation BSA release
Bovine Serum Albumin (BSA) was encapsulated into pH-responsive alginate/kappa(κ)-carrageenan double network hydrogel beads at different pHs and CaCl2/KCl ratios. Controlled release of BSA was performed in the simulated gastric fluid (SGF) and then in simulated intestinal fluid (SIF). Alginate reinforced the mechanical properties while double network with kappa-carrageenan made slow down the release of BSA into SIF. Sulfate groups on kappa-carrageenan improved the resistivity against phosphate buffer solution. Encapsulation and BSA release was manipulated by varying pH and crosslinking concentration. The highest encapsulation efficiency (83–89%) was obtained with the hydrogel beads prepared at the lower pH than isoelectric point of BSA, then 67–72% of them released in SIF in 4–5 h while lower than 10% of BSA released in SGF, hence, protein delivery targeted intestinal was achieved. BSA release exhibited a coupling of diffusion and polymer relaxation mechanism without burst release.
1. Introduction Therapeutic proteins have significant role in the treatment of many diseases, for example, human insulin is beneficial for diabetes, and nivolumab and nembrolizumab can be used for cancer immunotherapy [1,2]. However, there are many difficulties on the application of protein drugs like denaturation, aggregation, and hydrolyzation [3]. Therefore, efficacy, stability and biological activity of proteins can reduce when they are exposed to different environmental conditions such as humidity, temperature, pH, light, oxygen, gastrointestinal fluids. In order to overcome these problems, protein drugs can be encapsulated in a three dimensional, water or physiological fluid-swollen chemically or physically crosslinked macromolecular network, which is known as hydrogel. Synthetic and natural polymers are widely used forproducing hydrogels. Polysaccharide such as chitosan, alginate, carrageenan, hyaluronic acid, cellulose based hydrogels are gaining interest because of their great biodegradability and biocompatibility. Furthermore, there is more interest on drug delivery studies and effective drug delivery systems using polysaccharides [4,5]. Alginate is a natural anionic polysaccharide and biopolymer extracted from brown algae, and is comprised of varying quantity of 1,4′-linked β- D -mannuronic acid (M) and α-l-guluronic acid (G) monomers [6]. Alginate hydrogel is formed by physically crosslinking with divalent cations such as calcium ion, which is the most commonly
used because of its biocompatibility [7]. Alginate based hydrogels are extensively used as wound dressings in biomedical application [8], cell transplantation in tissue engineering and delivery of protein/drug in controlled release systems [9]. Another significant properties of alginate is pH sensitivity. pH dependence of alginate beads makes them suitable for designing oral delivery system. In gastric environment, alginate based hydrogel shrinks and cannot allow the entrapped bioactive proteins to release, but in intestinal environment, alginate beads become swollen so that encapsulated protein or drugs can be released [10]. Kappa carrageenan (κ-car) is a natural anionic polysaccharide, and linear sulphated biopolymer obtained from the tropical seaweed. It is composed of alternating 3-linked β-d-galactopyranose (G-units) and 4-linked α-d-galactopyranose (D-units) or 4-linked 3,6-anhydro-α-dgalactopyranose (DA-units) [11]. In biomedical applications, appropriate forms of the carrageenan based system such as beads, microcapsules and microspheres have been widely used to release of drugs or proteins because of its biocompability, biodegradability, the ability to form gel and thermo reversibility [12]. Gelation of κ-car takes place in two steps: in the first step, helix structure is formed at 80–90 C, and sulfate group of κ-car is neutralized by cations such as Ca+2, Na+ and K+, in the second step, three dimensional network is formed by decreasing the temperature to 50 °C [13]. It has been reported that stronger κ-car gels were obtained in the presence of KCl
Corresponding author. E-mail addresses: [email protected], [email protected] (D. Duranoğlu). 1 Present address: Gebze Technical University, Chemical Engineering Department, 41400, Gebze, Kocaeli, Turkey. ∗
https://doi.org/10.1016/j.jddst.2020.101551 Received 5 December 2019; Received in revised form 17 January 2020; Accepted 27 January 2020 Available online 30 January 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.
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compared to other salts such as NaCl, MgCl2, LiCl, SrCl2 and CaCl2 [14]. In particular, potassium ions promote the formation of an ionic bond with the sulfate group from one galactose residue and electrostatic interaction with an anhydrous-O-3,6-ring of another galactose residue, whereas divalent calcium ions are crosslinked with sulfate groups on neighboring single or double helices. These ion-mediated forces and intermolecular associations enhance chain stability [15,16]. Furthermore, gel–sol and sol–gel transition temperature is higher in the presence of potassium ions due to the lower electronegativity comparing to other monovalent ions (K+:0.82; Na+:0.93; Li+:0.98) [14]. The main purpose of this study is to synthesize biocompatible, mechanically strength, pH-resposive double crosslinked hydrogel networks in order to protect protein from acid hydrolyze and proteolytic breakdown passing through the gastrointestinal tract. Herein, polymer double network with a synergistic effects of alginate and κ-carrageenan was synthesized with a simple method. The effect of pH and the ratio of crosslinking agents (K+ and Ca2+ ions) on encapsulation efficiency of BSA, and on release behavior in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) was investigated comprehensively. Up to now, several studies have been published about the entrapment of betamethasone acetate [17], riboflavin [18], and α-amylase [19] in physically crosslinked alginate-κ-carrageenan hydrogel network. To the best of our knowledge, the effect of pH on encapsulation efficiency and BSA release profile of alginate/ĸ-carrageenan hydrogel bead has not been reported in the literature.
Loading capacity (mg/g) =
2.3. BSA release studies and release kinetics BSA release studies were performed in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF), respectively. 2 g of sodium chloride (NaCl) was dissolved in 500 mL of distilled water, then 7 mL of hydrochloric acid (37.4%) was added and the final solution was completed to 1 L with distilled water in order to prepare SGF (pH = 1.2) [16]. 6.8 g of potassium dihydrogen phosphate (KH2PO4) was dissolved in 500 mL of distilled water, then 190 mL of 0.2 M sodium hydroxide was added, and the final solution was completed to 1 L with distilled water in order to prepare SIF (pH = 7.4) [17]. BSA release experiments were carried with 5 g of BSA loaded wet beads placed in Erlenmeyer flask containing 100 mL of SGF. After 2 h stirring at 150 rpm at 37 °C, the beads were taken and filtered from SGF and transferred to 100 mL of SIF. 1mL sample was withdrawn from release medium at specified time intervals and filtered through a 0.45 μm membrane filter. BSA concentration was measured in release medium using UV spectrophotometry at 280 nm. Cumulative BSA release was calculated using the following Equation (3):
2.1. Materials
Cumulative Release (%) =
Bovine serum albumin (BSA) was purchased from Sigma-Aldrich. All other reagents have analytical grade and were purchased from Sigma-Aldrich. Sodium alginate (NaAlg) and Kappa-carrageenan (κ-car) were provided by Will Powder and Molecular Gastronomy Recipes, respectively.
Mt = kp × t n M
Mf
×100
(3)
(4)
where, n: diffusional exponent, which defines the mechanism of drug release; kp: rate constant; t: time.
NaAlg and κ-car aqueous dispersions were prepared separately. NaAlg powders (2% w/v) were dissolved in 70 mL of distilled water under constant stirring overnight. κ-car powders (2% w/v) were dissolved in 30 mL of distilled water, at 80 °C for an hour, then the temperature was reduced to 25 °C under constant stirring overnight. After 24 h, NaAlg and κ-car solutions were mixed together for 2 h with continuous stirring to obtain a uniform solution. 5 mL of 1% BSA solution was added to the NaAlg- κ-car mixture and pH of the mixture was adjusted to desired pH value by adding diluted HCl solution slowly. BSA loaded beads were formed by dropping the mixture into 250 mL of salt solution (%CaCl2:%KCl) using a peristaltic pump (Masterflex L/S; 5 mL/min flow rate; 16 cm tube diameter). The beads were kept in salt solution for 10 min for gelation and then diluted by 250 mL of distilled water. BSA loaded beads were filtrated from the salt solution and then kept in distilled water for 48 h for further usage. Schematic illustration of preperation of BSA loaded double network Alg-κ-car hydrogel beads can be seen in Fig. 1. In order to determine encapsulated BSA, BSA concentration was analyzed by using UV spectrophotometry (Analytic Jena Specord 200) at 280 nm. BSA encapsulation efficiency was calculated using the following Equation (1):
Mi
Mt ×100 M
where, Mt: the amount of BSA (mg) released after time t; M∞: the amount of BSA (mg) loaded in the beads. BSA release kinetic was analyzed by Ritger-Peppas Model [20]:
2.2. Preparation of BSA loaded Alg-κ-car beads
Mi
(2)
The FTIR–ATR spectra of the samples were obtained using a Bruker Optics Alpha-P, in the 400–4000 cm−1 region and at a resolution of 4 cm−1. Spectra were obtained by collecting the average of 20 scans. Surface morphology of the beads was observed by using Scanning Electron Microscope (SEM, Zeiss EVO LS 10). The beads were coated with gold and operated at an accelerating voltage of 5 kV.
2. Experimental
Encapsulation Efficiency (%) =
amount of encapsulated BSA (mg ) total amount of wet beads (g )
3. Results and discussion BSA loaded beads prepared with various CaCl2:KCl concentration, and pH are shown in Table 1 with the corresponding encapsulation efficiencies and loading capacities. 3.1. Characterization of hydrogel beads Surface morphology of hydrogel beads was observed by using SEM (Fig. 2). As can be seen that Alg-κ-car hydrogel beads (prepared with the 2% of CaCl2) have no large pores and present tight surface (Fig. 2a) although BSA loaded hydrogel bead (B7) has a rough surface (Fig. 2b). It was observed that BSA bound to bead structure and was trapped in the bead, however, accumulation of BSA on the surface was not observed clearly (Fig. 2b). Functional groups of BSA, hydrogel beads and BSA loaded hydrogel beads (B7) were determined by using FT-IR spectrometer (Fig. 3). FTIR-ATR spectrum of BSA loaded hydrogel bead (B7) almost overlapped with the spectrum of hydrogel bead. The characteristic peaks of BSA at 1639 and 1521 cm−1 which are due to C]O stretching vibrations of the amide I (-NH2) group and bond between N–H of amide II (-NH-) group [21] was not observed on the FTIR-ATR spectrum of BSA loaded hydrogel (Fig. 3).
(1)
where Mi: the amount of BSA (mg) added to NaAlg- κ-car mixture; Mf: the amount of BSA (mg) in salt solution. BSA loading capacity of hydrogels was calculated by using Equation (2): 2
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Fig. 1. Schematic illustration of preperation of BSA loaded Alg-κ-car hydrogel beads.
groups on the alginate molecules are available for cross-linking with calcium ions [26]. Higher amount of anionic groups on NaAlg and -κ-car at pH 7.5 could be resulted with the highest cross-linking, which can affect the BSA encapsulation of hydrogel beads in a positive way. It can be concluded that electrostatic attraction and higher crosslinking degree are dominant for BSA encapsulation on hydrogel beads prepared at pH below and above isoelectric point, respectively.
Table 1 BSA loaded beads. Formulation
B1 B2 B3 B4 B5 B6 B7 B8 B9
pH
4.2 4.2 4.2 4.8 4.8 4.8 7.5 7.5 7.5
Salt Concentration
Encapsulation Efficiency (EE)
BSA Loading Capacity
CaCl2:KCl (%:%)
(%)
(mg BSA/g bead)
2:0 2:2 4:4 2:0 2:2 4:4 2:0 2:2 4:4
83 85 89 29 36 53 62 64 68
18.4 18.4 18.6 5.5 7.1 10.9 11.7 12.2 15.4
3.3. Effect of crosslinking salt concentration on BSA encapsulation BSA encapsulation efficiency of hydrogel beads prepared at different cosslinking salt concentration ratios (CaCl2:KCl) was also investigated. Fig. 4 shows that the BSA encapsulation efficiency increases as the salt concentration ratio increases at each pH value (pH 4.2, 4.8 and 7.5). The encapsulation efficiency of BSA loaded beads prepared at pH 4.2 and pH 7.5 showed a slight increase with an increase in the salt concentration. Low encapsulation efficiency at low salt concentrations can be explained with the insufficient cross-linking. During bead formation, BSA can be diffused out of insufficient cross-linking beads. It has been reported that the BSA encapsulation efficiency of alginate-chitosan beads loaded with BSA increased by increasing CaCl2 concentration [27]. The increase in the encapsulation efficiency with the salt concentration of BSA loaded beads prepared at isoelectric point (pH 4.8) is much higher than that of the others. This can be explained that, due to the weak electrostatic attraction between polymer and BSA, with the increasing salt concentration, cross-linking becomes more effective on encapsulation efficiency at isoelectric point.
3.2. Effect of pH on BSA encapsulation BSA can electrostatically interact with the pH-sensitive alginate and κ-car biopolymers. pKa value of mannuronic and gluronic units of alginate are 3.38 and 3.65, respectively [18], and the pKa value of κ-car is about 2 [18,22]. Thus, electrostatic interaction between BSA and alginate-κ-car can be manipulated by varying the pH. The effect of pH, below (pH 4.2), above (pH 7.5) and at isoelectric point (pH 4.8), on encapsulation efficiency was investigated. BSA molecules have net zero charge at isoelectric point (pH 4.8) while positively charged at pH 4.2, and negatively charged at pH 7.5 [23]. BSA encapsulation efficiencies obtained at different pHs are shown in Fig. 4. As can be seen that the lowest encapsulation efficiency was obtained at pH 4.8 while the highest encapsulation efficiency was obtained at pH 4.2. This can be interpereted with the interactions of charges of both hydrogel beads and BSA. In the values below the isoelectric point of BSA, BSA molecules are positively charged while κ-car and NaAlg are negatively charged [24]. Therefore, encapsulation efficiency reached at the highest value (89%) due to the electrostatic attraction between negatively charged alginate and κ-car in the beads and positively charged BSA molecules at pH 4.2. Similarly, it has been reported that BSA loading efficiency was higher at lower pH values than isoelectric point of protein because of the electrostatic interaction between BSA and alginate [25]. At pH 7.5, encapsulation efficiency was obtained lower (68%) than that of pH 4.2 since the electrostatic repulsion is dominant between negatively charged BSA and ionized carboxylic groups in the hydrogel beads. However, encapsulation efficiency obtained at isoelectric point of BSA is the lowest (53%) due to the decreasing electrostatic attraction between ionized carboxylic and sulfonic groups in the beads and neutral BSA molecules. At the pH values above the isoelectric point of BSA, there must be more strong interaction than the electrostatic repulsion of negatively charged groups. As it is known that anionic
3.4. BSA release studies 3.4.1. BSA release in SGF BSA release diffusion from hydrogel beads is controlled by both the swelling behavior and mechanical strength of the hydrogel beads and the chemical structure of the protein. After BSA release performed in SGF for the first 2 h, hydrogel beads were transferred into SIF. pH sensitive hydrogel beads became smaller due to the protonation of carboxylic and sulfate groups of alginate-κ-car at pH 1.2, so that BSA diffusion from hydrogel beads was very low, then, cumulative BSA release in SGF was obtained less than 10% (Figs. 5–7). To our knowledge, except [28], there is no previous study reported lower than 10% of BSA release from alginate contained hydrogel beads into SGF (Table 4). We thought that the presence of sulfate groups in the κ-car was significantly effective on inhibition of BSA release in SGF. Sulfate groups in κ-car can be resulted in higher crosslinking, i.e., smaller pore size, so that inhibiting BSA release. Similarly, Xu et al. decreased the BSA release into SGF from about 80% to 3% by adding SO42− ions into hydrogel structure [29]. 3
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Fig. 2. SEM images of a) Alg-κ-car hydrogel beads b) BSA loaded hydrogel beads (B7).
3.4.3. Effect of synthesis pH on BSA release As can be seen from Fig. 5, BSA release reached 72%, 80%, 21% from B1, B4, B7 hydrogel beads, 61%; 84%; 27% from B2; B5; B8 hydrogel beads, and 67%, 70%, 22% from B3, B6, B9 hydrogel beads, respectively. Among all formulations, highest BSA release percentage was obtained with the hydrogel beads prepared at isoelectric point, due to the weak electrostatic interactions between BSA molecules and hydrogel structure. It was followed by hydrogel beads prepared at slightly acidic pH (4.2). The least BSA release percentage was observed with the hydrogel beads due to the highest crosslinking ratio prepared at basic media (pH 7.5). However, the highest amount (in mg/g) of BSA release was obtained from the hydrogel beads, which were prepared with the highest encapsulation efficiency at pH 4.2 (Table 2). Salt concentration has little effect on BSA release from the beads prepared at pH 4.2 and 7.5, whereas, it is more effective on BSA release from the beads prepared at pH 4.8.
3.4.2. BSA release in SIF As can be seen BSA release profiles in Figs. 5–7 that, BSA was gradually released from the hydrogel matrix into SIF in 2–4 h. In SIF (pH 7.4), calcium ions were replaced by potassium and sodium ions. This change led to an increase in the swelling potential of the hydrogel beads, at the same time decreasing the stability of the hydrogel beads, hence, the amount of BSA released in SIF was greater. The porosity of the hydrogel matrix, the solubility of BSA, the swelling capacity of the hydrogel matrix and erosion in the releasing environment can affect BSA release in SIF [24]. Anal et al. stated that due to the chelating effect of phosphate ions, the disruption of the calcium alginate matrix was faster in the phosphate buffer solution at above pH 5.5 [27]. Li et al. obtained 180 μg BSA release in SIF (pH: 7.5) while 11 g BSA release in SGF (pH: 1.2) from 3 mg chitosan-carrageenan polyelectrolyte complex [30]. In another study, the BSA protein loaded on alginate-N,O carboxymethyl chitosan hydrogel beads was released at approximately 82–87% at pH 7.5 [31]. In the study of Sevgi and Aybige, the entire BSA encapsulated in the alginate-chitosan beads was released in the phosphate buffer solution (pH; 6.8) in 1 h [32]. 4
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Fig. 3. FTIR-ATR spectra of BSA, hydrogel beads and BSA loaded hydrogel.
Fig. 6. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = %2:%2. Fig. 4. Effect of pH and CaCl2:KCl (%:%) ratio on BSA encapsulation efficiencies.
Fig. 7. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = 4:4. Fig. 5. Cumulative release profiles of BSA from Alg-κ-car hydrogel beads prepared with CaCl2:KCl = %2:%0.
According to the Ritger-Peppas model, each of the ln(Mt/M∞) versus ln(t) graphs produced linear curves. The correlation coefficients (0.913–0.995) indicated that chosen kinetic model succesfully fitted the experimental data (Table 3). The diffusional exponents (n) were obtained from the slope of the linear curves to verify the release mechanism of BSA from hydrogel beads. According to the Ritger and Peppas [20], for the swellable spherical matrices such as alginate-κ-car hydrogel beads, if the n value is equal or less than 0.43, there is a Case I (Fickian diffusion); if n is equal or greater than 0.85, case II (polymer
3.5. BSA release kinetics The release data of BSA from Alg-κ-car hydrogel beads into SIF medium were fitted to the Ritger-Peppas model and the release mechanism of BSA was investigated. Ritger-Peppas equation parameters and correlation coefficients were given in Table 3. 5
Journal of Drug Delivery Science and Technology 56 (2020) 101551
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in calcium and potassium crosslinked Alg-κ-car hydrogel beads at different pH and salt concentrations, was achieved by both the protein diffusion from beads and relaxation of hydrogel macromolecular structure. It was also observed during the period of BSA release in the SIF that the hydrogel beads first became swollen, very large and soft, and then lost their spherical structure by the time. Kaygusuz and Erim observed that 85% of BSA released from alginate hydrogel beads in 2 h [28], whereas, we observed the maximum 83% BSA release in 3 h (Fig. 6). It can be concluded that the burst release was inhibited, and the controlled release behavior of BSA was improved by using double network Alg-κ-car hydrogel beads in our study. This can be attributed to the highly crosslinked structure of prepared hydrogel as well as the resistivity against phosphate due to the sulfate groups on kappa-carrageenan.
Table 2 The results of BSA release study. Formulation
B1 B2 B3 B4 B5 B6 B7 B8 B9
Encapsulation Efficiency
Cumulative BSA release
Released BSA
BSA Release Yield*
(%)
(%)
(mg/g bead)
(%)
83 85 89 29 36 53 62 64 68
72 61 67 80 84 70 21 27 22
13.3 11.2 12.4 5.5 5.7 7.6 2.9 3.4 3.5
72 61 67 100 80 70 25 28 23
BSA Release Yield (%) =
the amount of released BSA (mg ) theinitial amount of BSA (mg )
4. Conclusion
× 100 .
pH-responsive double network alginate/kappa-carrageenan hydrogel beads were prepared by ion gelation method in order to encapsulate protein. BSA encapsulation was performed at three different pH values and different crosslinking salt solution concentrations. The highest BSA encapsulation efficiency (89%) was obtained with the B3 formulation, which was prepared with the highest cosslinking salt ratio (CaCl2:KCl = 4%:4%) at pH 4.2. The strong interaction between the positively charged BSA and negatively charged alginate and kappa carrageenan, and the high crosslinking salt concentration led to high encapsulation efficiency. BSA release from pH-responsive hydrogel beads in simulated gastric fluid (SGF) was negligible amount (less than 10%). In simulated intestine fluid (SIF), the highest BSA release occurred (13.3 mg/g) from the hydrogel beads prepared with the lowest crosslinking salt concentration (CaCl2:KCl = 2%:0%) at pH 4.2. According to Ritger-Peppas kinetic model, BSA release showed an anomalous release mechanism (diffusive behavior and polymer relaxation) i.e. BSA could release in intestine in a controlled manner. Preparing double network hydrogel beads with alginate and kappa-carrageenan as well as varying the synthesis pH and crosslinking salt concentration not only resulted with the high encapsulation efficiency and also controlled release of BSA in the intestine without burst release.
Table 3 Ritger-Peppas kinetic model parameters and correlation coefficients. Formulation
n
R2
B1 B2 B3 B4 B5 B6 B7 B8 B9 Alginate [28]
0.82 0.69 0.68 0.93 0.71 0.68 0.57 0.62 0.60 1.05
0.979 0.995 0.987 0.913 0.978 0.950 0.942 0.945 0.965 0.970
relaxation) transport mechanism dominates. In many cases, molecules release with a kinetic behavior that is dependent on the relative ratio of diffusion and relaxation mechanism, which is called as anomalous transport, where the diffusional exponent, n, is between the 0.43 < n˂0.85 [20]. According to obtained n values varying from 0.57 to 0.82 (Table 3), the release of the BSA from alginate-κ-car hydrogel beads exhibited both diffusion and polymer relaxation behavior. BSA diffusional exponents, n, obtained from the beads prepared at pH 4.2 and 4.8 decreased with the increasing salt concentration (n = 0.82 for B1; n = 0.68 for B3 and n = 0.93 for B4; n = 0.68 for B6). It can be explained that the stronger cross-linking by increasing the salt concentration inhibited the polymer relaxation, so diffusion was thought to be more effective than polymer relaxation for the high salt concentration. As a result, the release of BSA protein, which was loaded
CRediT authorship contribution statement Selin Sarıyer: Methodology, Investigation, Writing - original draft, Visualization. Dilek Duranoğlu: Supervision, Conceptualization, Methodology, Resources, Data curation, Writing - review & editing. Özlem Doğan: Conceptualization, Methodology, Data curation, Writing - review & editing, Funding acquisition. İlknur Küçük:
Table 4 BSA encapsulation and release in alginate based hydrogel beads. Hydrogel
Crosslinking agent
BSA Encapsulation Efficiency
BSA Release in SGF
BSA Release in SIF/PBS
Ref.
Alginate Alginate/P(CE-MAA-MEG) Alginate Alginate/N,O-carboxymethyl chitosan Alginate/Carboxymethyl Chitin Alginate Alginate/hydroxypropyl-methylcellulose Alginate/Poly(N-isopropyl acrylamide) Alginate/Chitosan
CaCl2 CaCl2 CaCl2 CaCl2 FeCl3 FeCl3 CaCl2·2H2O CaCl2 CaCl2 CaCl2/Na2SO4 CaCl2 CaCl2 BaCl2+ CaCl2 CaCl2 CaCl2 CaCl2/KCl
Not mentioned 72.3–78.6% 47–51% Not mentioned 95.4–96.8% 98.1% 69% 25% ≥97%
49.7% 13.4 Not experimented 25% 14–18% 15% Not experimented Not experimented ≥80% 1.76–2.35% 27% 9–13% Not experimented Not experimented Not experimented ≤10%
93.6% 60% 86-74% 80% 70% 80% 45% (pH = 7.1) 62% (pH = 7.4) 95% (pH = 6.8) 82.86–52.91% (pH = 6.8) 85% (pH = 7.2) 45–55% (pH = 7.2) 79.7% (pH = 7.4) 97.7% (pH = 7.4) 90% 67–72% 13.3–12.4 mg/g
[33]
Alginate Alginate/montmorillonite Alginate Alginate Alginate/Chitosan Alginate/Kappa-carrageenan
40% 78% 88% 86% Not mentioned 83–89% 18.4–18.6 mg/g
6
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