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Ionically crosslinked alginate–carboxymethyl cellulose beads for the delivery of protein therapeutics
Applied Surface Science 262 (2012) 28–33
Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Ionically crosslinked alginate–carboxymethyl cellulose beads for the delivery of protein therapeutics Min Sup Kim, Sang Jun Park, Bon Kang Gu, Chun-Ho Kim ∗ Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Science, Seoul 139-240, South Korea
a r t i c l e
i n f o
Article history: Available online 21 January 2012 Keywords: Alginate Carboxymethyl cellulose Ionic crosslinked beads Controlled release Oral protein delivery Tissue engineering
a b s t r a c t We developed Fe3+ -crosslinked alginate–carboxymethyl cellulose (AC) beads in various volume ratios by dropping an AC solution into a ferric chloride solution to form protein therapeutic carrier beads. Scanning electron microscopy revealed that the roughness and pore size of the crosslinked beads increased with the volume ratio of the carboxymethyl cellulose. Fourier transform-infrared analysis revealed the formation of a three-dimensional bonding structure between the anionic polymeric chains of AC and the Fe3+ ions. The degree of swelling and the release profile of albumin from the beads were investigated under simulated gastrointestinal conditions (pH 1.2, 4.5, and 7.4). The Fe3+ -crosslinked AC beads displayed different degrees of swelling and albumin release for the various AC volume ratios and under various pH conditions. An in vitro release test was used to monitor the controlled release of albumin from the AC beads under simulated gastrointestinal conditions over 24 h. The Fe3+ -crosslinked AC beads protected and controlled the release of protein, demonstrating that such beads present a promising protein therapeutic carrier for the oral delivery. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Protein therapeutic delivery systems provide controlled release to deliver a therapeutic to a targeted site and promptly achieve and maintain the desired local therapeutic concentration [1–3]. Oral delivery of protein therapeutics is the preferred method of administration because it is more comfortable and less invasive than injection delivery. However, several major problems are associated with oral delivery. Proteins are degraded by proteolytic enzymes and the acidic environment of the stomach. To mitigate these problems, polymeric beads have been developed based on natural polymers [4–6]. Alginate (AL) is a family of linear polysaccharides, a natural polymer, with mucoadhesive properties, low toxicity, and good biodegradability. These properties enable use of the beads for the encapsulation and delivery of therapeutic proteins. Alginate beads prepared by ionic crosslinking with Ca2+ have been investigated in the context of oral delivery [7–11]. Carboxymethyl cellulose (CMC), a water-soluble polysaccharide prepared by the chemical modification of cellulose, also has tremendous potential for use in site-specific or controlled-release drug delivery systems, due to its high biocompatibility, biodegradability, and low immunogenicity [12–14].
∗ Corresponding author at: Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Science, 215-4, Gongneung-dong, Nowon-gu, Seoul 139706, Korea. Tel.: +82 2 970 1319; fax: +82 2 970 1317. E-mail address: [email protected] (C.-H. Kim).
Many studies have examined drug release from Fe3+ -crosslinked CMC-based hydrogels as protein carriers [15–17]. Oral delivery of protein drugs requires that the protein be protected from the harsh environment in the stomach. However, Fe3+ -crosslinked CMC-based hydrogels are unstable in acid media, leading to early release or degradation of the drug [18]. Protein drugs delivered in a CMC matrix are, therefore, poorly absorbed in the intestine due to degradation by proteolytic enzymes in the gastrointestinal tract. Additionally, protein drugs frequently suffer from poor membrane permeability, which further reduces their absorption. Recently, bio-active molecules, including cytokines, insulin, liposome, and cell-penetrating functional peptides, were incorporated into polysaccharide-based hydrogels, which guarantee protein drug activity and improve the delivery of protein drugs via the oral route [19,20]. To reduce protein release from protein carriers in the gastric environment, hydrogels composed of alginate and carboxymethyl cellulose (AC) were prepared in the form of beads by dropping aqueous AC solutions into a ferric chloride solution. The preparation process is straightforward, relatively fast, and occurs in an all-aqueous environment. High temperatures and the use of organic solvents, which may degrade or denature the protein, are avoided [4,21]. In this study, we fabricated Fe3+ -crosslinked AC beads for the delivery of a model protein, albumin. The goal was to enhance the stability of the beads and to control the release of albumin in the gastrointestinal environment. Variations in the size, morphology, and chemical integrity of the crosslinked AC beads were
0169-4332/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.01.010
M.S. Kim et al. / Applied Surface Science 262 (2012) 28–33
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investigated. Additionally, the degree of swelling in the AC beads was evaluated in pH 1.2, 4.5, and 7.4 media. Finally, we observed the release profile of albumin from the AC beads under gastrointestinal conditions over 24 h. 2. Materials and methods 2.1. Materials AL (sodium salt, from brown algae), CMC (sodium salt, average MW, 250 kDa), ferric chloride, and fluorescein isothiocyanateconjugated albumin (from bovine, BSA-FITC) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium chloride (KCl) and hydrochloric acid (HCl) were also purchased from Sigma–Aldrich. Dulbecco’s phosphate buffered saline (PBS) was purchased from Gibco BRL (Carlsbad, CA, USA). 2.2. Preparation of ionically crosslinked beads AL and CMC were separately dissolved in deionized (DI) water to form 2% (w/v) stock solutions. A series of AL-CMC blends with stock solution volume ratios of AL:CMC (AC) of 2:0, 2:1, 1:1, 1:2,and 0:2 (AL2, A2C1, A1C1, A1C2, and CMC2, respectively) were prepared by thorough stirring until the solution became viscous and homogeneous. Following the polymeric solution dropping method, the resulting solutions were injected from a stainless steel needle connected to a syringe using a syringe pump (KD scientific, Holliston, MA, USA) into a 2% (w/v) ferric chloride solution at 5 mL/h. The ferric chloride solution was placed 10 cm below the tip of the needle. After the crosslinking reaction proceeded for 30 min, the beads were rinsed with DI water three times to remove unreacted Fe3+ . Subsequently, the beads were lyophilized at −80 ◦ C in a vacuum (5 mTorr) over 1 day. After lyophilization, the morphology of each bead was characterized using a MIRA II field-emission scanning electron microscope (FE-SEM; Tescan, Libusinatr 21, Czech Republic). 2.3. Viscosity of the polymeric solutions The viscosity of a 2% (w/v) AC solution was measured using an AR 2000 EX rheometer (TA instrument, New Castle, DE, USA) with a cone and plate geometry 4 cm in diameter and a 1◦ cone angle. Each solution was subjected to rotations of the cone and plate to induce controlled shear rate. The viscosities of the solutions were measured at 20 ◦ C for 5 min under a constant shear stress of 1 Pa. 2.4. Fourier transform infrared analysis The chemical integrity of the ionically crosslinked beads was investigated by Fourier transform infrared (FTIR) spectroscopy (Tensor 27; Bruker Optics, Ettlingen, Germany). Five milligrams of each bead were cut into small pieces to prepare KBr discs, and FTIR spectra over the range 4000–500 cm−1 were recorded. 2.5. Degree of swelling in the ionically crosslinked beads The degree of swelling in the beads was determined in vitro by measuring changes in the weight of the samples exposed to simulated gastrointestinal conditions, including solutions containing HCl–KCl (pH 1.2, mixed with 0.2 M HCl and 0.2 M KCl at volume ratio of 1.7:1), HCl–KCl–PBS (pH 4.5, PBS added into HCl–KCl), and PBS (pH 7.4) [19]. The initial dry weight of each bead (W1) was determined. After the weighing process, beads were incubated and agitated in each pH buffer at 37 ◦ C. After 3 h, the swollen beads were collected and weighed (W2), and the percentage of the initial
Fig. 1. Viscosities of the 2% (w/v) alginate, carboxymethyl cellulose, and blended solutions. (The viscosity of each solution was determined at 20 ◦ C with a constant shear stress of 1 Pa.)
weight was calculated according to the following equation; degree of swelling in the beads (%) = 100 × [(W2 − W1)/W1]. 2.6. Preparation of ionically crosslinked beads containing BSA-FITC Beads containing BSA-FITC were prepared by dissolving 1 mg/mL BSA-FITC in an AC solution containing AL:CMC volume ratios of 2:0, 2:1, 1:1, 1:2, and 0:2 (AL2, A2C1, A1C1, A1C2 and CMC2, respectively). After mixing BSA-FITC with the polymeric solution, ionically crosslinked beads were fabricated as described above. To investigate the encapsulation efficiency of BSA-FITC in beads, each group of beads (50 mg) was crushed and incubated in 10 mL PBS at 37 ◦ C for 24 h, and the mixtures were centrifuged at 3000 rpm. The amount of BSA-FITC in the supernatant was determined by measuring the optical density (OD) at 490 nm using a plate reader (Spectra Max M2e; Molecular Devices, Sunnyvale, CA, USA). A standard calibration curve was prepared using known concentrations of the BSA-FITC solution. 2.7. BSA-FITC release experiment The release kinetics of BSA-FITC from beads was determined by incubating samples in 10 mL media at pH 1.2, pH 4.5, and pH 7.4 at 37 ◦ C for 24 h with continuous agitation. Samples (100 L) were collected at 0, 1, 3, 6, 12, and 24 h. The cumulative amount of released BSA-FITC in the collected solution was determined from OD measurements, as described above. The release profiles of BSA-FITC under the pH conditions of digestion were measured by changing the pH of the medium from acidic to neutral. The initial pH of 1.2 during the first 3 h was increased to pH 7.4 for the subsequent 21 h. At 0, 1, 3, 6, 12, and 24 h, the amount of BSA-FITC released was determined as described above. 2.8. Statistical analysis Quantitative data were obtained in triplicate and are reported as means ± standard deviations, where indicated. Statistical analysis was performed using a one-way analysis of variation (ANOVA), followed by the Tukey HSD for multiple comparisons. A p-value <0.05 was considered statistically significant.
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Fig. 2. Representative SEM images of (A) AL2, (B) A2C1, (C) A1C1, (D) A1C2, and (E) CMC2 beads. The scale bars in the large and small inset indicate 500 m and 100 m.
We measured the viscosities of 2% (w/v) AC solutions prepared in AL:CMC ratios of 2:0, 2:1, 1:1, 1:2,and 0:2. As shown in Fig. 1, the viscosity of the AL2 solution was 306.39 ± 1.22 mPa s, and the viscosity of the AC solution decreased as the volume ratio of CMC increased, to 262.65 ± 1.61 mPa s (A2C1), 244.48 ± 0.85 mPa s (A1C1), 225.66 ± 0.76 mPa s (A1C2), and 185.95 ± 0.42 mPa s (CMC2). These results indicated that the viscosity of the AC solution could be controlled by varying the volume ratio of the AC solution.
and 1.7 ± 0.1 mm (CMC2). Moreover, the surface roughness of the beads increased with increasing CMC. Next, we observed the crosssection of Fe3+ -crosslinked AC beads by FE-SEM, as shown in Fig. 3. The cross-sectional images of all AC beads showed core-shell structures, and the inner cores had highly porous structures with non-woven microfibers. SEM images of the A1C2 bead revealed partial cleavage through sections of the beads. The cleavage induced the formation of void structures and micropores in the CMC2 beads. The morphological differences between the A1C2 and CMC2 beads suggested that the higher diffusion rate of the CMC2 solution (due to the lower viscosity of the CMC solution) produced rough surfaces on the beads and larger aggregates of hydrophilic polymer chains during the crosslinking process [14,22,23].
3.2. Morphology of the Fe3+ -crosslinked AC beads
3.3. FTIR analysis of Fe3+ -crosslinked AC beads
As shown in Fig. 2, the Fe3+ -crosslinked AC beads were observed by SEM. The diameter of the beads increased slightly by increasing the amount of CMC in the volume ratio to 1.3 ± 0.2 mm (AC2)
FTIR spectra of the raw materials and crosslinked beads, presented in Fig. 4(A and B) show a broad hydroxyl band near 3400 cm−1 in all samples, which was attributable to an OH
3. Results and discussion 3.1. Viscosity of AC solutions
Fig. 3. Cross-sectional views of (A) AL2, (B) A2C1, (C) A1C1, (D) A1C2, and (E) CMC2 beads. The scale bars in the large and small insets indicate 50 m and 10 m.
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Fig. 5. Degree of swelling in Fe3+ -crosslinked beads at pH 1.2, 4.5, and 7.4 at 37 ◦ C for 3 h.
CMC2 beads was observed at 1740 cm−1 , possibly due to electrostatic interactions between the carboxyl groups of AL and CMC with Fe3+ [26]. The carboxyl group peak was also observed in the crosslinked A2C1, A1C1, and A1C2 beads of Fig. 4(C).
3.4. Degree of swelling in the AC beads under various pH conditions The degree of swelling in ionically crosslinked anionic polymer hydrogels containing carboxylic groups decreased under acidic conditions due to formation of hydrogen bonds between the COOH and OH groups. However, under neutral conditions, the carboxyl groups were partially ionized and electrostatic repulsion increased the degree of swelling [27–29]. As shown in Fig. 5, the degree of swelling in the Fe3+ -crosslinked AC beads was measured under simulated gastrointestinal pH conditions (pH 1.2, 4.5, and 7.4). At pH 7.4, the degree of swelling of the AL2 beads increased dramatically to 108.62 ± 5.61% compared to 28.71 ± 3.72% and 41.72 ± 2.72% at pH 1.2 and pH 4.5, respectively. However, the degree of swelling in Fe3+ -crosslinked beads was significantly lower than that of the calcium–alginate reported by Lee et al. [30]. Because calcium cation is divalent, their bonding to alginate occurs in a planar two-dimensional manner, as illustrated in the egg–box model [31]. In contrast, the trivalent cation, Fe3+ , is expected to form a three-dimensional bonding structure. Extended three-dimensional crosslinking decreased water uptake by the beads [17]. However, at pH 1.2, the degree of swelling (59.52 ± 2.21%) of the CMC2 beads was higher than in other samples. These trends were observed at pH 4.5, and the degree of swelling (95.15 ± 2.16%) in CMC2 beads was higher than in other blend beads at pH 7.4. The high degree of swelling in the CMC2 beads under acidic and neutral conditions is related to the presence of micropores in the inner core, as shown in Fig. 3(E).
3.5. Encapsulation efficiency of BSA-FITC in the AC beads Fig. 4. FTIR spectra of (A) AL powder, and AL2 beads, (B) CMC powder and CMC2 beads, (C) A2C1, A1C1, and A1C2 beads.
stretching mode. In these spectra, the peaks near 1620 cm−1 and 1420 cm−1 were attributed to the asymmetrical and symmetrical vibrations of the carboxylate ions on AL and CMC [24]. The broad bands over the range 1200–1000 cm−1 were attributed to sugar ring absorption [25]. A new small peak in the crosslinked AL2 and
We investigated the encapsulation efficiency of BSA-FITC in the AC beads after the crosslinking and washing processes, as shown in Table 1. The encapsulation efficiency of BSA-FITC by AL2 beads was 96.12 ± 2.21%, slightly lower than 87.76 ± 3.85%, observed in beads with a higher volume ratio of CMC2. These results suggested that the three-dimensional bonding structure of the Fe3+ -crosslinked AC beads resulted in the higher encapsulation efficiency for BSA-FITC.
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M.S. Kim et al. / Applied Surface Science 262 (2012) 28–33
Table 1 Encapsulated Efficiency of BSA-FITC in Fe3+ crosslinked AC beads. Code name
Concentration of BSA-FITC (mg/mL)
Efficiency of encapsulation (%)
AL2 A2C1 A1C1 A1C2 CMC2
1 1 1 1 1
91.12 85.99 84.63 75.21 67.76
± ± ± ± ±
2.21 2.22 3.26 2.24 3.85
3.6. Release profile of BSA-FITC from AC beads The release of BSA-FITC from Fe3+ -crosslinked AC beads was measured under pH 1.2 and pH 7.4 conditions. Fig. 6(A) shows the release profiles of BSA-FITC under pH 1.2 conditions, in which CMC2 beads with micropores exhibit a fast release phase, amounting to 8.96 ± 1.52% and 19.84 ± 3.35% BSA-FITC at 3 and 6 h, respectively. The BSA-FITC release from AL2 beads was comparatively slow, 3.46 ± 1.33% and 7.41 ± 0.85% of the total encapsulated BSAFITC. The other samples (A2C1, A1C1, and A1C2) showed similar BSA-FITC release behavior. The release behavior could be explained as follows. The carboxyl groups of the alginate were protonated at pH 1.2, and a low degree of swelling limited diffusion of BSAFITC [27]. Fig. 6(B) shows the release behavior of BSA-FITC from AC beads at pH 7.4. CMC2 beads showed an initial burst of BSAFITC release, amounting to 40.97 ± 1.73% and 90.72 ± 4.91% of the initially encapsulated BSA-FITC at 1 and 3 h, respectively. There-
Fig. 7. Release behavior of BSA-FITC from Fe3+ -crosslinked beads in pH 1.2 media over 3 h then in pH 7.4 media over 21 h at 37 ◦ C.
after, a slight release of BSA-FITC was observed. However, BSA-FITC encapsulated in the A2C1, A1C1, and A1C2 beads was released over the course of 24 h. For example, BSA-FITC in A2C1, A1C1, and A1C2 beads was released at rates of 8.86 ± 2.28%, 6.48 ± 3.63%, and 5.87 ± 2.49% at 1 h, 38.77 ± 7.65%, 36.52 ± 8.26%, and 23.49 ± 3.76% at 6 h, and 54.75 ± 5.85%, 49.34 ± 10.61%, and 42.66 ± 3.12% at 12 h, respectively. The release rate of BSA-FITC from AL2 beads was faster than from A2C1, A1C1, and A1C2 beads due to the higher degree of swelling at pH 7.4. Finally, we investigated the release behavior of BSA-FITC from AC beads in the gastrointestinal environment by increasing the media pH from 1.2 to 7.4, as shown in Fig. 7. Under pH 1.2 conditions, a large amount of BSA-FITC (22.19 ± 3.19%) was released from the CMC2 beads over 3 h due to the presence of cleaved structures. The other beads showed a slow release of BSA-FITC, and the amount of BSA-FITC released did not exceed 5%. These results suggest that AL2, A2C1, A1C1, and A1C2 beads provided the desired protective effects for the oral delivery of proteins, because almost all encapsulated protein remained in the beads during passage through the low pH environment of the stomach. After transferred from acid to higher pH, a relatively slow release of BSA-FITC from AL2, A2C1, A1C1, and A1C2 beads was found compared to 66.21 ± 6.95% for AL2 and 74.83 ± 7.95% for CMC2 beads at 6 h. For example, BSA-FITC in A2C1, A1C1, and A1C2 beads was released at rates of 48.15 ± 7.73%, 42.81 ± 5.81%, and 45.61 ± 6.03% at 6 h, 72.52 ± 5.51%, 72.31 ± 9.21%, and 57.99 ± 6.13% at 12 h, and 84.19 ± 5.17%, 88.95 ± 2.39%, and 69.85 ± 4.13% at 24 h, respectively. The A1C2 beads provided a controlled release of proteins, and drug release over longer periods of time could be achieved in the intestines.
4. Conclusions
Fig. 6. Release behavior of BSA-FITC from the Fe3+ -crosslinked beads in (A) pH 1.2 and (B) pH 7.4 media at 37 ◦ C for 24 h.
In this study, we developed Fe3+ -crosslinked AC carrier beads by dropping an AC solution into a ferric chloride solution containing a protein therapeutic. The main advantage of this system is that all procedures used for preparing the beads and loading the proteins were performed in aqueous media, which is beneficial for preserving the bioactivity and stability of fragile proteins or drugs. A comparison with AL2 and CMC2 beads showed that the blend beads were more effective at controlling the release of protein therapeutics. Our bead system will be useful for bypassing the acidity of gastric fluids without wasting loaded protein or retarding
M.S. Kim et al. / Applied Surface Science 262 (2012) 28–33
protein release in the intestine. Such beads may be effective as a site-specific protein oral delivery system for the colon. Acknowledgments This work was supported by Nuclear Research Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by Ministry of Education, Science and Technology (MEST, Korea) (grant code: 2011-0002361 and 2011-0020757). References [1] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004) 1818. [2] R. Langer, New methods of drug delivery, Science 249 (1990) 1527. [3] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene delivery to cells and tissue, Adv. Drug Deliv. Rev. 55 (2003) 329–347. [4] N. Peppas, Devices based on intelligent biopolymers for oral protein delivery, Int. J. Pharm. 277 (2004) 11–17. [5] V. Sinha, A. Trehan, Biodegradable microspheres for protein delivery, J. Controlled Release 90 (2003) 261–280. [6] A. Vila, A. Sanchez, M. Tobi’o, P. Calvo, M. Alonso, Design of biodegradable particles for protein delivery, J. Controlled Release 78 (2002) 15–24. [7] W.R. Gombotz, S.F. Wee, Protein release from alginate matrices, Adv. Drug Deliv. Rev. 31 (1998) 267–285. [8] F. Gu, B. Amsden, R. Neufeld, Sustained delivery of vascular endothelial growth factor with alginate beads, J. Controlled Release 96 (2004) 463–472. [9] T. Gründer, J. Fritz, R. Stoop, P. Hortschansky, J. Mollenhauer, W.K. Aicher, Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads* 1, Osteoarthritis Cartilage 12 (2004) 559–567. [10] T.H. Tran, Y. Lee, K.M. Huh, Biofunctional polymeric nanoparticles for targeted drug delivery, Int. J. Tissue Regen. 1 (2010) 55–67. [11] O.Y. Kim, S.J. Kim, Y.S. Song, E.H. Jo, J.H. Hwang, J.Y. Bae, I.S. Yoo, D. Lee, K.H. Yoon, G. Khang, Purification and characterization of alginate for the application of regenerative medicine, Int. J. Tissue Regen. 2 (2011) 13–20. [12] A.P. Rokhade, S.A. Agnihotri, S.A. Patil, N.N. Mallikarjuna, P.V. Kulkarni, T.M. Aminabhavi, Semi-interpenetrating polymer network microspheres of gelatin and sodium carboxymethyl cellulose for controlled release of ketorolac tromethamine, Carbohydr. Polym. 65 (2006) 243–252. [13] Z. Liu, Y. Jiao, Y. Wang, C. Zhou, Z. Zhang, Polysaccharides-based nanoparticles as drug delivery systems, Adv. Drug Deliv. Rev. 60 (2008) 1650–1662. [14] H. Jo, D.S. Kim, M.S. Hong, H.L. Kim, W.H. Cho, D. Lee, G. Khang, Influence of the viscosity properties on drug release rate from hydroxymethylcellulose
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Contents lists available at SciVerse ScienceDirect
Applied Surface Science journal homepage: www.elsevier.com/locate/apsusc
Ionically crosslinked alginate–carboxymethyl cellulose beads for the delivery of protein therapeutics Min Sup Kim, Sang Jun Park, Bon Kang Gu, Chun-Ho Kim ∗ Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Science, Seoul 139-240, South Korea
a r t i c l e
i n f o
Article history: Available online 21 January 2012 Keywords: Alginate Carboxymethyl cellulose Ionic crosslinked beads Controlled release Oral protein delivery Tissue engineering
a b s t r a c t We developed Fe3+ -crosslinked alginate–carboxymethyl cellulose (AC) beads in various volume ratios by dropping an AC solution into a ferric chloride solution to form protein therapeutic carrier beads. Scanning electron microscopy revealed that the roughness and pore size of the crosslinked beads increased with the volume ratio of the carboxymethyl cellulose. Fourier transform-infrared analysis revealed the formation of a three-dimensional bonding structure between the anionic polymeric chains of AC and the Fe3+ ions. The degree of swelling and the release profile of albumin from the beads were investigated under simulated gastrointestinal conditions (pH 1.2, 4.5, and 7.4). The Fe3+ -crosslinked AC beads displayed different degrees of swelling and albumin release for the various AC volume ratios and under various pH conditions. An in vitro release test was used to monitor the controlled release of albumin from the AC beads under simulated gastrointestinal conditions over 24 h. The Fe3+ -crosslinked AC beads protected and controlled the release of protein, demonstrating that such beads present a promising protein therapeutic carrier for the oral delivery. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved.
1. Introduction Protein therapeutic delivery systems provide controlled release to deliver a therapeutic to a targeted site and promptly achieve and maintain the desired local therapeutic concentration [1–3]. Oral delivery of protein therapeutics is the preferred method of administration because it is more comfortable and less invasive than injection delivery. However, several major problems are associated with oral delivery. Proteins are degraded by proteolytic enzymes and the acidic environment of the stomach. To mitigate these problems, polymeric beads have been developed based on natural polymers [4–6]. Alginate (AL) is a family of linear polysaccharides, a natural polymer, with mucoadhesive properties, low toxicity, and good biodegradability. These properties enable use of the beads for the encapsulation and delivery of therapeutic proteins. Alginate beads prepared by ionic crosslinking with Ca2+ have been investigated in the context of oral delivery [7–11]. Carboxymethyl cellulose (CMC), a water-soluble polysaccharide prepared by the chemical modification of cellulose, also has tremendous potential for use in site-specific or controlled-release drug delivery systems, due to its high biocompatibility, biodegradability, and low immunogenicity [12–14].
∗ Corresponding author at: Laboratory of Tissue Engineering, Korea Institute of Radiological and Medical Science, 215-4, Gongneung-dong, Nowon-gu, Seoul 139706, Korea. Tel.: +82 2 970 1319; fax: +82 2 970 1317. E-mail address: [email protected] (C.-H. Kim).
Many studies have examined drug release from Fe3+ -crosslinked CMC-based hydrogels as protein carriers [15–17]. Oral delivery of protein drugs requires that the protein be protected from the harsh environment in the stomach. However, Fe3+ -crosslinked CMC-based hydrogels are unstable in acid media, leading to early release or degradation of the drug [18]. Protein drugs delivered in a CMC matrix are, therefore, poorly absorbed in the intestine due to degradation by proteolytic enzymes in the gastrointestinal tract. Additionally, protein drugs frequently suffer from poor membrane permeability, which further reduces their absorption. Recently, bio-active molecules, including cytokines, insulin, liposome, and cell-penetrating functional peptides, were incorporated into polysaccharide-based hydrogels, which guarantee protein drug activity and improve the delivery of protein drugs via the oral route [19,20]. To reduce protein release from protein carriers in the gastric environment, hydrogels composed of alginate and carboxymethyl cellulose (AC) were prepared in the form of beads by dropping aqueous AC solutions into a ferric chloride solution. The preparation process is straightforward, relatively fast, and occurs in an all-aqueous environment. High temperatures and the use of organic solvents, which may degrade or denature the protein, are avoided [4,21]. In this study, we fabricated Fe3+ -crosslinked AC beads for the delivery of a model protein, albumin. The goal was to enhance the stability of the beads and to control the release of albumin in the gastrointestinal environment. Variations in the size, morphology, and chemical integrity of the crosslinked AC beads were
0169-4332/$ – see front matter. Crown Copyright © 2012 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.apsusc.2012.01.010
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investigated. Additionally, the degree of swelling in the AC beads was evaluated in pH 1.2, 4.5, and 7.4 media. Finally, we observed the release profile of albumin from the AC beads under gastrointestinal conditions over 24 h. 2. Materials and methods 2.1. Materials AL (sodium salt, from brown algae), CMC (sodium salt, average MW, 250 kDa), ferric chloride, and fluorescein isothiocyanateconjugated albumin (from bovine, BSA-FITC) were purchased from Sigma–Aldrich (St. Louis, MO, USA). Potassium chloride (KCl) and hydrochloric acid (HCl) were also purchased from Sigma–Aldrich. Dulbecco’s phosphate buffered saline (PBS) was purchased from Gibco BRL (Carlsbad, CA, USA). 2.2. Preparation of ionically crosslinked beads AL and CMC were separately dissolved in deionized (DI) water to form 2% (w/v) stock solutions. A series of AL-CMC blends with stock solution volume ratios of AL:CMC (AC) of 2:0, 2:1, 1:1, 1:2,and 0:2 (AL2, A2C1, A1C1, A1C2, and CMC2, respectively) were prepared by thorough stirring until the solution became viscous and homogeneous. Following the polymeric solution dropping method, the resulting solutions were injected from a stainless steel needle connected to a syringe using a syringe pump (KD scientific, Holliston, MA, USA) into a 2% (w/v) ferric chloride solution at 5 mL/h. The ferric chloride solution was placed 10 cm below the tip of the needle. After the crosslinking reaction proceeded for 30 min, the beads were rinsed with DI water three times to remove unreacted Fe3+ . Subsequently, the beads were lyophilized at −80 ◦ C in a vacuum (5 mTorr) over 1 day. After lyophilization, the morphology of each bead was characterized using a MIRA II field-emission scanning electron microscope (FE-SEM; Tescan, Libusinatr 21, Czech Republic). 2.3. Viscosity of the polymeric solutions The viscosity of a 2% (w/v) AC solution was measured using an AR 2000 EX rheometer (TA instrument, New Castle, DE, USA) with a cone and plate geometry 4 cm in diameter and a 1◦ cone angle. Each solution was subjected to rotations of the cone and plate to induce controlled shear rate. The viscosities of the solutions were measured at 20 ◦ C for 5 min under a constant shear stress of 1 Pa. 2.4. Fourier transform infrared analysis The chemical integrity of the ionically crosslinked beads was investigated by Fourier transform infrared (FTIR) spectroscopy (Tensor 27; Bruker Optics, Ettlingen, Germany). Five milligrams of each bead were cut into small pieces to prepare KBr discs, and FTIR spectra over the range 4000–500 cm−1 were recorded. 2.5. Degree of swelling in the ionically crosslinked beads The degree of swelling in the beads was determined in vitro by measuring changes in the weight of the samples exposed to simulated gastrointestinal conditions, including solutions containing HCl–KCl (pH 1.2, mixed with 0.2 M HCl and 0.2 M KCl at volume ratio of 1.7:1), HCl–KCl–PBS (pH 4.5, PBS added into HCl–KCl), and PBS (pH 7.4) [19]. The initial dry weight of each bead (W1) was determined. After the weighing process, beads were incubated and agitated in each pH buffer at 37 ◦ C. After 3 h, the swollen beads were collected and weighed (W2), and the percentage of the initial
Fig. 1. Viscosities of the 2% (w/v) alginate, carboxymethyl cellulose, and blended solutions. (The viscosity of each solution was determined at 20 ◦ C with a constant shear stress of 1 Pa.)
weight was calculated according to the following equation; degree of swelling in the beads (%) = 100 × [(W2 − W1)/W1]. 2.6. Preparation of ionically crosslinked beads containing BSA-FITC Beads containing BSA-FITC were prepared by dissolving 1 mg/mL BSA-FITC in an AC solution containing AL:CMC volume ratios of 2:0, 2:1, 1:1, 1:2, and 0:2 (AL2, A2C1, A1C1, A1C2 and CMC2, respectively). After mixing BSA-FITC with the polymeric solution, ionically crosslinked beads were fabricated as described above. To investigate the encapsulation efficiency of BSA-FITC in beads, each group of beads (50 mg) was crushed and incubated in 10 mL PBS at 37 ◦ C for 24 h, and the mixtures were centrifuged at 3000 rpm. The amount of BSA-FITC in the supernatant was determined by measuring the optical density (OD) at 490 nm using a plate reader (Spectra Max M2e; Molecular Devices, Sunnyvale, CA, USA). A standard calibration curve was prepared using known concentrations of the BSA-FITC solution. 2.7. BSA-FITC release experiment The release kinetics of BSA-FITC from beads was determined by incubating samples in 10 mL media at pH 1.2, pH 4.5, and pH 7.4 at 37 ◦ C for 24 h with continuous agitation. Samples (100 L) were collected at 0, 1, 3, 6, 12, and 24 h. The cumulative amount of released BSA-FITC in the collected solution was determined from OD measurements, as described above. The release profiles of BSA-FITC under the pH conditions of digestion were measured by changing the pH of the medium from acidic to neutral. The initial pH of 1.2 during the first 3 h was increased to pH 7.4 for the subsequent 21 h. At 0, 1, 3, 6, 12, and 24 h, the amount of BSA-FITC released was determined as described above. 2.8. Statistical analysis Quantitative data were obtained in triplicate and are reported as means ± standard deviations, where indicated. Statistical analysis was performed using a one-way analysis of variation (ANOVA), followed by the Tukey HSD for multiple comparisons. A p-value <0.05 was considered statistically significant.
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Fig. 2. Representative SEM images of (A) AL2, (B) A2C1, (C) A1C1, (D) A1C2, and (E) CMC2 beads. The scale bars in the large and small inset indicate 500 m and 100 m.
We measured the viscosities of 2% (w/v) AC solutions prepared in AL:CMC ratios of 2:0, 2:1, 1:1, 1:2,and 0:2. As shown in Fig. 1, the viscosity of the AL2 solution was 306.39 ± 1.22 mPa s, and the viscosity of the AC solution decreased as the volume ratio of CMC increased, to 262.65 ± 1.61 mPa s (A2C1), 244.48 ± 0.85 mPa s (A1C1), 225.66 ± 0.76 mPa s (A1C2), and 185.95 ± 0.42 mPa s (CMC2). These results indicated that the viscosity of the AC solution could be controlled by varying the volume ratio of the AC solution.
and 1.7 ± 0.1 mm (CMC2). Moreover, the surface roughness of the beads increased with increasing CMC. Next, we observed the crosssection of Fe3+ -crosslinked AC beads by FE-SEM, as shown in Fig. 3. The cross-sectional images of all AC beads showed core-shell structures, and the inner cores had highly porous structures with non-woven microfibers. SEM images of the A1C2 bead revealed partial cleavage through sections of the beads. The cleavage induced the formation of void structures and micropores in the CMC2 beads. The morphological differences between the A1C2 and CMC2 beads suggested that the higher diffusion rate of the CMC2 solution (due to the lower viscosity of the CMC solution) produced rough surfaces on the beads and larger aggregates of hydrophilic polymer chains during the crosslinking process [14,22,23].
3.2. Morphology of the Fe3+ -crosslinked AC beads
3.3. FTIR analysis of Fe3+ -crosslinked AC beads
As shown in Fig. 2, the Fe3+ -crosslinked AC beads were observed by SEM. The diameter of the beads increased slightly by increasing the amount of CMC in the volume ratio to 1.3 ± 0.2 mm (AC2)
FTIR spectra of the raw materials and crosslinked beads, presented in Fig. 4(A and B) show a broad hydroxyl band near 3400 cm−1 in all samples, which was attributable to an OH
3. Results and discussion 3.1. Viscosity of AC solutions
Fig. 3. Cross-sectional views of (A) AL2, (B) A2C1, (C) A1C1, (D) A1C2, and (E) CMC2 beads. The scale bars in the large and small insets indicate 50 m and 10 m.
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Fig. 5. Degree of swelling in Fe3+ -crosslinked beads at pH 1.2, 4.5, and 7.4 at 37 ◦ C for 3 h.
CMC2 beads was observed at 1740 cm−1 , possibly due to electrostatic interactions between the carboxyl groups of AL and CMC with Fe3+ [26]. The carboxyl group peak was also observed in the crosslinked A2C1, A1C1, and A1C2 beads of Fig. 4(C).
3.4. Degree of swelling in the AC beads under various pH conditions The degree of swelling in ionically crosslinked anionic polymer hydrogels containing carboxylic groups decreased under acidic conditions due to formation of hydrogen bonds between the COOH and OH groups. However, under neutral conditions, the carboxyl groups were partially ionized and electrostatic repulsion increased the degree of swelling [27–29]. As shown in Fig. 5, the degree of swelling in the Fe3+ -crosslinked AC beads was measured under simulated gastrointestinal pH conditions (pH 1.2, 4.5, and 7.4). At pH 7.4, the degree of swelling of the AL2 beads increased dramatically to 108.62 ± 5.61% compared to 28.71 ± 3.72% and 41.72 ± 2.72% at pH 1.2 and pH 4.5, respectively. However, the degree of swelling in Fe3+ -crosslinked beads was significantly lower than that of the calcium–alginate reported by Lee et al. [30]. Because calcium cation is divalent, their bonding to alginate occurs in a planar two-dimensional manner, as illustrated in the egg–box model [31]. In contrast, the trivalent cation, Fe3+ , is expected to form a three-dimensional bonding structure. Extended three-dimensional crosslinking decreased water uptake by the beads [17]. However, at pH 1.2, the degree of swelling (59.52 ± 2.21%) of the CMC2 beads was higher than in other samples. These trends were observed at pH 4.5, and the degree of swelling (95.15 ± 2.16%) in CMC2 beads was higher than in other blend beads at pH 7.4. The high degree of swelling in the CMC2 beads under acidic and neutral conditions is related to the presence of micropores in the inner core, as shown in Fig. 3(E).
3.5. Encapsulation efficiency of BSA-FITC in the AC beads Fig. 4. FTIR spectra of (A) AL powder, and AL2 beads, (B) CMC powder and CMC2 beads, (C) A2C1, A1C1, and A1C2 beads.
stretching mode. In these spectra, the peaks near 1620 cm−1 and 1420 cm−1 were attributed to the asymmetrical and symmetrical vibrations of the carboxylate ions on AL and CMC [24]. The broad bands over the range 1200–1000 cm−1 were attributed to sugar ring absorption [25]. A new small peak in the crosslinked AL2 and
We investigated the encapsulation efficiency of BSA-FITC in the AC beads after the crosslinking and washing processes, as shown in Table 1. The encapsulation efficiency of BSA-FITC by AL2 beads was 96.12 ± 2.21%, slightly lower than 87.76 ± 3.85%, observed in beads with a higher volume ratio of CMC2. These results suggested that the three-dimensional bonding structure of the Fe3+ -crosslinked AC beads resulted in the higher encapsulation efficiency for BSA-FITC.
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Table 1 Encapsulated Efficiency of BSA-FITC in Fe3+ crosslinked AC beads. Code name
Concentration of BSA-FITC (mg/mL)
Efficiency of encapsulation (%)
AL2 A2C1 A1C1 A1C2 CMC2
1 1 1 1 1
91.12 85.99 84.63 75.21 67.76
± ± ± ± ±
2.21 2.22 3.26 2.24 3.85
3.6. Release profile of BSA-FITC from AC beads The release of BSA-FITC from Fe3+ -crosslinked AC beads was measured under pH 1.2 and pH 7.4 conditions. Fig. 6(A) shows the release profiles of BSA-FITC under pH 1.2 conditions, in which CMC2 beads with micropores exhibit a fast release phase, amounting to 8.96 ± 1.52% and 19.84 ± 3.35% BSA-FITC at 3 and 6 h, respectively. The BSA-FITC release from AL2 beads was comparatively slow, 3.46 ± 1.33% and 7.41 ± 0.85% of the total encapsulated BSAFITC. The other samples (A2C1, A1C1, and A1C2) showed similar BSA-FITC release behavior. The release behavior could be explained as follows. The carboxyl groups of the alginate were protonated at pH 1.2, and a low degree of swelling limited diffusion of BSAFITC [27]. Fig. 6(B) shows the release behavior of BSA-FITC from AC beads at pH 7.4. CMC2 beads showed an initial burst of BSAFITC release, amounting to 40.97 ± 1.73% and 90.72 ± 4.91% of the initially encapsulated BSA-FITC at 1 and 3 h, respectively. There-
Fig. 7. Release behavior of BSA-FITC from Fe3+ -crosslinked beads in pH 1.2 media over 3 h then in pH 7.4 media over 21 h at 37 ◦ C.
after, a slight release of BSA-FITC was observed. However, BSA-FITC encapsulated in the A2C1, A1C1, and A1C2 beads was released over the course of 24 h. For example, BSA-FITC in A2C1, A1C1, and A1C2 beads was released at rates of 8.86 ± 2.28%, 6.48 ± 3.63%, and 5.87 ± 2.49% at 1 h, 38.77 ± 7.65%, 36.52 ± 8.26%, and 23.49 ± 3.76% at 6 h, and 54.75 ± 5.85%, 49.34 ± 10.61%, and 42.66 ± 3.12% at 12 h, respectively. The release rate of BSA-FITC from AL2 beads was faster than from A2C1, A1C1, and A1C2 beads due to the higher degree of swelling at pH 7.4. Finally, we investigated the release behavior of BSA-FITC from AC beads in the gastrointestinal environment by increasing the media pH from 1.2 to 7.4, as shown in Fig. 7. Under pH 1.2 conditions, a large amount of BSA-FITC (22.19 ± 3.19%) was released from the CMC2 beads over 3 h due to the presence of cleaved structures. The other beads showed a slow release of BSA-FITC, and the amount of BSA-FITC released did not exceed 5%. These results suggest that AL2, A2C1, A1C1, and A1C2 beads provided the desired protective effects for the oral delivery of proteins, because almost all encapsulated protein remained in the beads during passage through the low pH environment of the stomach. After transferred from acid to higher pH, a relatively slow release of BSA-FITC from AL2, A2C1, A1C1, and A1C2 beads was found compared to 66.21 ± 6.95% for AL2 and 74.83 ± 7.95% for CMC2 beads at 6 h. For example, BSA-FITC in A2C1, A1C1, and A1C2 beads was released at rates of 48.15 ± 7.73%, 42.81 ± 5.81%, and 45.61 ± 6.03% at 6 h, 72.52 ± 5.51%, 72.31 ± 9.21%, and 57.99 ± 6.13% at 12 h, and 84.19 ± 5.17%, 88.95 ± 2.39%, and 69.85 ± 4.13% at 24 h, respectively. The A1C2 beads provided a controlled release of proteins, and drug release over longer periods of time could be achieved in the intestines.
4. Conclusions
Fig. 6. Release behavior of BSA-FITC from the Fe3+ -crosslinked beads in (A) pH 1.2 and (B) pH 7.4 media at 37 ◦ C for 24 h.
In this study, we developed Fe3+ -crosslinked AC carrier beads by dropping an AC solution into a ferric chloride solution containing a protein therapeutic. The main advantage of this system is that all procedures used for preparing the beads and loading the proteins were performed in aqueous media, which is beneficial for preserving the bioactivity and stability of fragile proteins or drugs. A comparison with AL2 and CMC2 beads showed that the blend beads were more effective at controlling the release of protein therapeutics. Our bead system will be useful for bypassing the acidity of gastric fluids without wasting loaded protein or retarding
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protein release in the intestine. Such beads may be effective as a site-specific protein oral delivery system for the colon. Acknowledgments This work was supported by Nuclear Research Development Program of the Korea Science and Engineering Foundation (KOSEF) grant funded by Ministry of Education, Science and Technology (MEST, Korea) (grant code: 2011-0002361 and 2011-0020757). References [1] T.M. Allen, P.R. Cullis, Drug delivery systems: entering the mainstream, Science 303 (2004) 1818. [2] R. Langer, New methods of drug delivery, Science 249 (1990) 1527. [3] J. Panyam, V. Labhasetwar, Biodegradable nanoparticles for drug and gene delivery to cells and tissue, Adv. Drug Deliv. Rev. 55 (2003) 329–347. [4] N. Peppas, Devices based on intelligent biopolymers for oral protein delivery, Int. J. Pharm. 277 (2004) 11–17. [5] V. Sinha, A. Trehan, Biodegradable microspheres for protein delivery, J. Controlled Release 90 (2003) 261–280. [6] A. Vila, A. Sanchez, M. Tobi’o, P. Calvo, M. Alonso, Design of biodegradable particles for protein delivery, J. Controlled Release 78 (2002) 15–24. [7] W.R. Gombotz, S.F. Wee, Protein release from alginate matrices, Adv. Drug Deliv. Rev. 31 (1998) 267–285. [8] F. Gu, B. Amsden, R. Neufeld, Sustained delivery of vascular endothelial growth factor with alginate beads, J. Controlled Release 96 (2004) 463–472. [9] T. Gründer, J. Fritz, R. Stoop, P. Hortschansky, J. Mollenhauer, W.K. Aicher, Bone morphogenetic protein (BMP)-2 enhances the expression of type II collagen and aggrecan in chondrocytes embedded in alginate beads* 1, Osteoarthritis Cartilage 12 (2004) 559–567. [10] T.H. Tran, Y. Lee, K.M. Huh, Biofunctional polymeric nanoparticles for targeted drug delivery, Int. J. Tissue Regen. 1 (2010) 55–67. [11] O.Y. Kim, S.J. Kim, Y.S. Song, E.H. Jo, J.H. Hwang, J.Y. Bae, I.S. Yoo, D. Lee, K.H. Yoon, G. Khang, Purification and characterization of alginate for the application of regenerative medicine, Int. J. Tissue Regen. 2 (2011) 13–20. [12] A.P. Rokhade, S.A. Agnihotri, S.A. Patil, N.N. Mallikarjuna, P.V. Kulkarni, T.M. Aminabhavi, Semi-interpenetrating polymer network microspheres of gelatin and sodium carboxymethyl cellulose for controlled release of ketorolac tromethamine, Carbohydr. Polym. 65 (2006) 243–252. [13] Z. Liu, Y. Jiao, Y. Wang, C. Zhou, Z. Zhang, Polysaccharides-based nanoparticles as drug delivery systems, Adv. Drug Deliv. Rev. 60 (2008) 1650–1662. [14] H. Jo, D.S. Kim, M.S. Hong, H.L. Kim, W.H. Cho, D. Lee, G. Khang, Influence of the viscosity properties on drug release rate from hydroxymethylcellulose
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