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Albumin-crosslinked alginate hydrogels as sustained drug release carrier
Materials Science and Engineering C 27 (2007) 870 – 874 www.elsevier.com/locate/msec
Albumin-crosslinked alginate hydrogels as sustained drug release carrier Daisuke Tada, Toshizumi Tanabe ⁎, Akira Tachibana, Kiyoshi Yamauchi Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Received 11 May 2006; received in revised form 19 September 2006; accepted 10 October 2006 Available online 13 November 2006
Abstract To take advantage of the drug-binding ability of albumin as a component of drug delivery system, we have prepared hydrogels consisting of alginic acid (AL) and recombinant human serum albumin (rHSA) by dehydrating condensation using N-hydroxysuccininimide and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide. As rHSA content increased, the swelling ratio of the hydrogel decreased, indicating rHSA functioned as a crosslinker. In fact, trypsin treatment solubilized the hydrogel. Salicylic acid, which has high affinity for rHSA, was loaded most on the hydrogel of the highest rHSA content despite the lowest swelling ratio. Meanwhile, drugs with less affinity for HSA such as o-anisic acid and benzoic acid were preferably loaded on the hydrogel having the highest swelling ratio but the lowest HSA content. The release of salicylic acid from the hydrogel sustained longer than o-anisic acid and benzoic acid, reflecting the affinity of the drug for HSA. Furthermore, the hydrogel could carry much of positively charged dibucaine by the interaction with anionic alginic acid and showed highly sustained release. Since the safety of AL and rHSA in medical use is guaranteed, rHSA-crosslinked AL hydrogel is expected to use as a sustained drug release carrier for drugs having affinity for HSA and those with cationic charge. © 2006 Elsevier B.V. All rights reserved. Keywords: Albumin; Biodegradable hydrogel; Drug delivery; Sustained release; Crosslinking with proteins; Alginic acid
1. Introduction It is important to supply drugs to the patient in a controlled rate enabling the appropriate concentration of drugs and prolonged effectiveness. Crosslinked hydrogel networks have been investigated as controlled drug delivery systems, taking advantage of their functions to up-take and release drugs. Drugs confined in a hydrogel polymer network are released to a milieu in a rate controlled by the swelling behavior of hydrogels, the pore size of a polymer network, the affinity between drugs and hydrogelconstituting polymers, and hydrogel degradation in vivo. Hydrogels consisting of ionic polymers such as hydroxyethyl methacrylate and methacrylic acid have been extensively applied to the drug delivery vehicle due to the ionic interaction between polymers and counterionic drugs and the swelling behavior varying with pH and buffer conditions. [1–8] However, these hydrogels are not degradable by either hydrolytic or enzymatic mechanisms, which limit their potentials as biodegradable drug carriers. ⁎ Corresponding author. Tel.: +81 6 6605 3094; fax: +81 6 6605 2785. E-mail address: [email protected] (T. Tanabe). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.10.008
Recently, we prepared a hydrogel in which bovine serum albumin (BSA) was used as nodes of three-dimensional hydrogel network by linking BSA with poly(acryl amide) chains, focusing on the binding ability of albumin to various substances such as amino acids and drugs. [9] Usage of albumin as a crosslinking molecule has advantages in preventing albumin from leakage out of a hydrogel, which may occur if albumin is merely embedded in a hydrogel. In addition, the hydrogel was shown to become solubilized by proteolytic digestion of BSA, indicating that the hydrogel would be cleared in the body, which may decrease harmful body response caused by semipermanent residual of a hydrogel in the body. [10] BSAcrosslinked poly(acryl amide) hydrogel was demonstrated to show sustained release for drugs having affinity for albumin, that is, the drugs with high affinity to BSA was loaded in a higher amount and was released more slowly than those having less affinity to BSA. However, acryl amide monomer is likely to be harmful if it remained in a hydrogel and BSA possibly causes immunogenic adverse effects for human. Therefore, we have replaced BSA and acryl amide with recombinant human serum albumin (rHSA) [11] and alginic acid (AL), respectively. Since
D. Tada et al. / Materials Science and Engineering C 27 (2007) 870–874
HSA and AL have been granted as pharmaceuticals or foods additive, respectively, the safety of resultant hydrogel is guaranteed. Since AL is anionic polysaccharide, the resultant hydrogel is expected to bind to both drugs with cationic charge and drugs having affinity to albumin. The present paper describes the preparation of hydrogels consisting of rHSA and AL and their in vitro evaluation as a drug carrier.
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2.3. Swelling of hydrogels
2. Materials and methods
The hydrogel was left in distilled water until reaching to a constant size. Then, the weight of the hydrogel was measured after the moisture on the surface was removed with a filter paper. The hydrogel was also weighed after drying for a week in a desiccator. The swelling ratios (SRs) of hydrogels were calculated using SR = (Ws − Wd) / Wd, where Ws and Wd are the weight of the swollen and dried hydrogels, respectively.
2.1. Materials
2.4. Trypsin treatment of HSA-AL and EDA-AL hydrogels
Recombinant human serum albumin (rHSA) was kindly provided from Mitsubishi Pharma Corporation (Osaka). Sodium alginate 80–120 cP (AL), ethylenediamine (EDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and salicylic acid were purchased from Wako Pure Chemical Industries (Osaka). Sodium benzoate, o-anisic acid, and dibucaine hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The hydrogels swollen in distilled water were immersed in Dulbecco's phosphate buffered saline (-) (DPBS) containing 0.25%(w/v) of trypsin at 37 °C for 2 weeks, changing trypsin solution everyday. The change in hydrogel appearance was recorded with photographs.
2.2. Preparation of rHSA-crosslinked alginate hydrogel (HSA-AL) AL was dissolved in 0.05 M 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.6) and cooled to 4 °C on ice. EDC and NHS were added to AL solution to activate carboxylic acid groups of AL. After 20-minutes preactivation at 4 °C, rHSAwas added to the reaction mixture with gentle shaking. A combined weight of rHSA and AL was 600 mg and the volume of the mixture was adjusted to 12 ml with MES buffer. The amount of rHSA and AL actually used in hydrogel preparation is shown in Table 1. Then the reaction mixture was cast to perfluoro alkoxy fluoroplastics (PFA) dish (internal diameter: 50 mm) on which glass plate was placed to avoid drying and stood for 1 day at 4 °C. The resultant hydrogel was cut to a cylindrical shape by cork borer (diameter: 8.5 mm). Thus obtained hydrogel disk was washed in 50 g/l of Na2HPO4 solution and distilled water at 4 °C for 3 days, respectively. The hydrogel disk was stored in distilled water at 4 °C. The EDA-crosslinked AL hydrogels (EDA-AL) were prepared as a control in a similar manner to the HSA-AL. The compositions of the EDA-AL are also shown in Table 1.
Table 1 Preparation of hydrogels and their swelling ratios Hydrogel
HSA or EDA (mg)
HSA-AL (11:1) HSA-AL (5:1) HSA-AL (1:1) HSA-AL (1:5) EDA-AL (1:33) EDA-AL (1:1000)
550 500 300 100 18 0.6
a b c
AL (mg)
AL-COOH/HSA or EDA a (mol/mol)
Swelling ratio b (Ws/Wd) c
50 100 300 500 582 599
30 67 330 1670 10 320
44 ± 7 56 ± 7 81 ± 4 137 ± 14 41 ± 3 122 ± 18
The mol ratio of monosaccharide in AL to HSA or EDA. Each hydrogel came to equilibrium in distilled water. Ws and Wd are the weights of the swollen and dried hydrogels, respectively.
2.5. Adsorption of drugs to rHSA Twenty milliliters of rHSA aqueous solution (5 mg/ml) was placed in a seamless cellulose tubing (molecular cutoff of 12,000–16,000; Wako Pure Chemical Industries) and immersed in 380 ml of 0.53 mM drug solution at room temperature for 3 days, by which the drug concentration reached to a constant value. Then, the amount of the drug adsorbed to one rHSA molecule was calculated from the drug concentration in the outer solution. 2.6. Drug loading on a hydrogel Each drug was dissolved in distilled water to prepare 2 mM solution. Hydrogels swollen in distilled water were soaked in each drug solution at room temperature for 3 days. Then, the drug-loaded hydrogels were dried for 1 week in a desiccator. 2.7. Drug release from hydrogels The dried drug-loaded hydrogels (10 mg) were immersed in 50 ml of DPBS at room temperature. The medium was periodically replaced, and the amount of drug released into the medium was quantified by measuring the absorbance of the drug at 296 nm (salicylic acid and o-anisic acid in 1 M HCl), 224 nm (sodium benzoate), or 246 nm (dibucaine hydrochloride in 1 M HCl). After 2 weeks, the hydrogels were placed in DPBS at 60 °C for 24 h to complete the drug release. UV spectra were collected using a U-2010 spectrophotometer (Hitachi High Technologies, Tokyo). 3. Results and discussion 3.1. Hydrogel preparation Hydrogels consisting of rHSA and AL were prepared according to the procedure shown in Fig. 1. AL was firstly converted its activated ester by dehydrating condensation with NHS by the aid of water-soluble carbodiimide, EDC. Subsequently,
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Fig. 1. Preparation of HSA-AL hydrogel.
AL chains were crosslinked with HSA by the reaction of amino residues in HSA with activated ester groups in AL. Alginate hydrogels crosslinked with ethylene diamine (EDA) were similarly prepared as a control. The amount of HSA and AL used in hydrogels preparation and swelling ratio of resultant hydrogels are shown in Table 1. The increase of HSA or EDA feeding ratio to AL lowered the swelling ratio of a resultant hydrogel. HSA-AL hydrogels gradually swelled and reached to dissolution by the treatment with trypsin, while EDA-AL hydrogels retained their shape by the 2-weeks trypsin treatment (data not shown). Thus, HSA-AL hydrogel would gradually be cleared by the proteolysis of HSA in the body. 3.2. Drug loading HSA-AL hydrogel was loaded with salicylic acid, o-anisic acid, benzoic acid and dibucaine hydrochloride by immersing the hydrogel in each drug solution and subsequent drying. Table 2 shows the change in drug concentration before and after the hydrogel immersion into the drug solution. The data of o-anisic
acid were not shown in Table 2, since those were identical with those of benzoic acid. The concentration of dibucaine was heavily decreased by immersion of both HSA-and EDA-AL hydrogels and the decrease was more eminent for hydrogels of higher AL content, suggesting that the hydrogels adsorbed dibucaine through the ionic interaction of its positive charge with anionic AL. Among the three negatively charged drugs, only salicylic acid concentration slightly decreased when hydrogels of higher HSA content were used. This result suggests that salicylic acid was loaded on HSA-AL hydrogel by active adsorption of salicylic acid to HSA in addition to simple diffusion of the drug into a hydrogel. Then, these four drugs were briefly evaluated for the affinity to HSA by the method described in Materials and Methods. As shown in Fig. 2, the number of drugs bound to one HSA was determined to be 14 for salicylic acid, 5 for o-anisic acid, 3 for benzoic acid and 4 for dibucaine hydrochloride, supporting the results of drug loading. Although o-anisic acid and benzoic acid showed slight binding to rHSA, it might not be enough to change drug concentration upon drug loading.
Table 2 The drug concentration before and after the hydrogel immersion Hydrogel
HSA-AL (11:1) HSA-AL (5:1) HSA-AL (1:1) HSA-AL (1:5) EDA-AL (1:33) EDA-AL (1:1000)
Salicylic acid
Benzoic acid
Dibucaine
Before After (mM) (mM)
Before After (mM) (mM)
Before After (mM) (mM)
1.94 1.94 1.94 1.94 1.94
1.91 ± 0.01 1.92 ± 0.01 1.94 ± 0.00 1.94 ± 0.00 1.94 ± 0.00
1.92 1.92 1.92 1.94 1.94
1.91 1.91 1.91 1.91 1.91
1.90 ± 0.00 1.87 ± 0.01 1.75 ± 0.01 1.73 ± 0.02 1.50 ± 0.03
1.94
1.94 ± 0.00 1.94
1.94 ± 0.00 1.91
1.60 ± 0.01
1.94 ± 0.00 1.94 ± 0.00 1.94 ± 0.01 1.94 ± 0.00 1.94 ± 0.00
Fig. 2. Number of drug molecules adsorbed to one rHSA molecule. n = 3.
D. Tada et al. / Materials Science and Engineering C 27 (2007) 870–874 Table 3 Total amount of drug released from each hydrogel Hydrogel
Swelling Total release amount (μmol/10 mg gel) ratio a (Ws/Wd) b Salicylic o-Anisic Benzoic Dibucaine acid acid acid hydrochloride
HSA-AL (11:1) 44 ± 7 HSA-AL (5:1) 56 ± 7 HSA-AL (1:1) 81 ± 4 HSA-AL (1:5) 137 ± 14 EDA-AL (1:33) 41 ± 3 EDA-AL 122 ± 18 (1:1000) a b
2.99 ± 0.22 1.90 ± 0.12 0.75 ± 0.02 1.36 ± 0.03 0.37 ± 0.01 1.01 ± 0.03
0.78 ± 0.05 0.47 ± 0.05 0.63 ± 0.03 1.88 ± 0.04 0.48 ± 0.01 1.51 ± 0.01
0.66 ± 0.02 0.65 ± 0.01 0.80 ± 0.02 1.52 ± 0.08 0.45 ± 0.02 1.04 ± 0.08
3.5 ± 0.2 5.6 ± 0.2 16.1 ± 0.3 23.1 ± 2.8 29.0 ± 1.3 35.9 ± 0.3
Each hydrogel was left to swell in distilled water. Ws and Wd are the weights of the swollen and dried hydrogels, respectively.
3.3. Drug Release The drug release from each hydrogel was carried out by immersing the dried drug-loaded hydrogel in PBS at room temperature. Table 3 shows the total amount of drug released from a hydrogel, which might correspond to the amount of a drug loaded on a hydrogel, since further drug release was not observed when the hydrogel was incubated at 60 °C for 24 h after the drug release was ceased at room temperature. The amount of released dibucaine increased according to the increase of AL content in a hydrogel both for HSA-AL and EDA-AL hydrogels, reflecting the increase of ionic interaction between a cationic drug and anionic AL. When o-anisic acid and benzoic acid were loaded on HSA-AL hydrogels, the
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highest drug release was observed for HSA-AL (1:5) hydrogel, probably because of its highest swelling ratio. Although the release of these drugs decreased for HSA-AL (1:1) and (5:1) in a decreasing order of swelling ratio, the release slightly increased for HSA-AL (11:1) which had the lowest swelling ratio but the highest HSA content. This result is in good accordance with Fig. 2 indicating that both o-anisic acid and benzoic acid have slight binding ability to HSA. In contrast, salicylic acid was released most when loaded on HSA-AL (11:1) hydrogel and its release gradually decreased according to the decrease of HSA content up to 1:1 of HSA-AL ratio in spite of the increase of swelling ratio. These results coincide with those obtained in drug loading, indicating that salicylic acid was loaded on a hydrogel of higher HSA content mainly through binding to HSA, reflecting the high affinity of salicylic acid for HSA. Salicylic acid release was again raised when loaded on HSA-AL (1:5) hydrogel having highest swelling ratio, in which loading occur mainly through simple diffusion of the drug into a hydrogel along hydrogel swelling. Fig. 3 shows the time course of the release of each drug from HSA-AL hydrogels. As shown in Fig. 3a, salicylic acid release continued even after 24 h for HSA-AL (11:1) hydrogel and was completed within 4 h for HSA-AL (1:5) hydrogel. The release from HSA-AL (5:1) and (1:1) hydrogels were ceased around 10 h and 6 h, respectively. Thus, HSA-AL hydrogel of higher HSA content carried higher amount of salicylic acid and its release continued longer because of the binding to HSA. Benzoic acid and o-anisic acid release from HSA-AL (11:1), (5:1) and (1:1) hydrogels retained for about 5 h, while the release from HSA-AL (1:5) hydrogel
Fig. 3. Cumulative drug release from each hydrogels: (a) salicylic acid, (b) benzoic acid, (c) o-anisic acid and (d) dibucaine chloride. Symbols: closed circles, HSA-AL (11:1); closed triangles, HSA-AL (5:1); closed squares, HSA-AL (1:1), closed diamonds, HSA-AL (1:5), open circles, EDA-AL (1:33), and open diamonds, EDA-AL (1:1000). n = 3.
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ceased within 2 h (Fig. 3b and c), which was similar to that from EDA-AL hydrogels. These results also indicate that binding of benzoic acid and o-anisic acid to HSA took place to some extent in hydrogels of high HSA content. Release of dibucaine continued longer irrelevantly to HSA content of hydrogel, because of strong electrostatic interaction with AL (Fig. 3d). Thus, hydrogels consisting of recombinant HSA and AL have been prepared and evaluated as a drug carrier. Since HSA and AL have been granted as pharmaceuticals or foods additive, their safety is guaranteed. When HSA-AL hydrogel is administered to the body, the hydrogel would be solubilized by the proteolysis of HSA and resultant alginic acid would be excreted out of the body as demonstrated by uptake of alginic acid as foods. [12] From the present results showing sustained drug release for cationic drugs and the drugs having HSA affinity of HSA-AL hydrogel, HSA-AL hydrogel is expected to use as a safe sustained drug release carrier. Acknowledgement The authors are grateful to Mitsubishi Pharma Corporation for providing recombinant human serum albumin.
References [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12]
S.W. Kim, Y.H. Bae, T. Okano, Pharm. Res. 9 (1992) 283. A.R. Khare, N.A. Peppas, Biomaterials 16 (1995) 559. A.R. Khare, N.A. Peppas, J. Biomater. Sci., Polym. Ed. 4 (1993) 275. A. Gutowska, J.S. Bark, I.C. Kwon, Y.H. Bae, Y. Cha, S.W. Kim, J. Control. Release 48 (1997) 141. P.M. de la Torre, Y. Enobakhare, G. Torrado, S. Torrado, Biomaterials 24 (2003) 1499. S. Torrado, P. Prada, P.M. de la Torre, S. Torrado, Biomaterials 25 (2004) 917. M. Şen, C. Uzun, O. Güven, Int. J. Pharm. 203 (2000) 149. C. Alvarez-Lorenzo, A. Concheiro, J. Control. Release 80 (2002) 247. D. Tada, T. Tanabe, A. Tachibana, K. Yamauchi, J. Biosci. Bioeng. 100 (2005) 551. L.H. Christensen, V.B. Breiting, A. Aasted, A. Jørgensen, A.I. Kebuladze, Plast. Reconstr. Surg. 111 (2003) 1883. K. Kobayashi, N. Nakamura, A. Sumi, T. Ohmura, K. Yokoyama, Ther. Apher. 2 (1998) 257. A.S. Sandberg, H. Anderson, I. Bosaeus, N.G. Carlsson, K. Hasselblad, M. Harrod, Am. J. Clin. Nutr. 60 (1994) 751.
Albumin-crosslinked alginate hydrogels as sustained drug release carrier Daisuke Tada, Toshizumi Tanabe ⁎, Akira Tachibana, Kiyoshi Yamauchi Department of Applied Chemistry and Bioengineering, Graduate School of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan Received 11 May 2006; received in revised form 19 September 2006; accepted 10 October 2006 Available online 13 November 2006
Abstract To take advantage of the drug-binding ability of albumin as a component of drug delivery system, we have prepared hydrogels consisting of alginic acid (AL) and recombinant human serum albumin (rHSA) by dehydrating condensation using N-hydroxysuccininimide and 1-ethyl-3-(3dimethylaminopropyl)carbodiimide. As rHSA content increased, the swelling ratio of the hydrogel decreased, indicating rHSA functioned as a crosslinker. In fact, trypsin treatment solubilized the hydrogel. Salicylic acid, which has high affinity for rHSA, was loaded most on the hydrogel of the highest rHSA content despite the lowest swelling ratio. Meanwhile, drugs with less affinity for HSA such as o-anisic acid and benzoic acid were preferably loaded on the hydrogel having the highest swelling ratio but the lowest HSA content. The release of salicylic acid from the hydrogel sustained longer than o-anisic acid and benzoic acid, reflecting the affinity of the drug for HSA. Furthermore, the hydrogel could carry much of positively charged dibucaine by the interaction with anionic alginic acid and showed highly sustained release. Since the safety of AL and rHSA in medical use is guaranteed, rHSA-crosslinked AL hydrogel is expected to use as a sustained drug release carrier for drugs having affinity for HSA and those with cationic charge. © 2006 Elsevier B.V. All rights reserved. Keywords: Albumin; Biodegradable hydrogel; Drug delivery; Sustained release; Crosslinking with proteins; Alginic acid
1. Introduction It is important to supply drugs to the patient in a controlled rate enabling the appropriate concentration of drugs and prolonged effectiveness. Crosslinked hydrogel networks have been investigated as controlled drug delivery systems, taking advantage of their functions to up-take and release drugs. Drugs confined in a hydrogel polymer network are released to a milieu in a rate controlled by the swelling behavior of hydrogels, the pore size of a polymer network, the affinity between drugs and hydrogelconstituting polymers, and hydrogel degradation in vivo. Hydrogels consisting of ionic polymers such as hydroxyethyl methacrylate and methacrylic acid have been extensively applied to the drug delivery vehicle due to the ionic interaction between polymers and counterionic drugs and the swelling behavior varying with pH and buffer conditions. [1–8] However, these hydrogels are not degradable by either hydrolytic or enzymatic mechanisms, which limit their potentials as biodegradable drug carriers. ⁎ Corresponding author. Tel.: +81 6 6605 3094; fax: +81 6 6605 2785. E-mail address: [email protected] (T. Tanabe). 0928-4931/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2006.10.008
Recently, we prepared a hydrogel in which bovine serum albumin (BSA) was used as nodes of three-dimensional hydrogel network by linking BSA with poly(acryl amide) chains, focusing on the binding ability of albumin to various substances such as amino acids and drugs. [9] Usage of albumin as a crosslinking molecule has advantages in preventing albumin from leakage out of a hydrogel, which may occur if albumin is merely embedded in a hydrogel. In addition, the hydrogel was shown to become solubilized by proteolytic digestion of BSA, indicating that the hydrogel would be cleared in the body, which may decrease harmful body response caused by semipermanent residual of a hydrogel in the body. [10] BSAcrosslinked poly(acryl amide) hydrogel was demonstrated to show sustained release for drugs having affinity for albumin, that is, the drugs with high affinity to BSA was loaded in a higher amount and was released more slowly than those having less affinity to BSA. However, acryl amide monomer is likely to be harmful if it remained in a hydrogel and BSA possibly causes immunogenic adverse effects for human. Therefore, we have replaced BSA and acryl amide with recombinant human serum albumin (rHSA) [11] and alginic acid (AL), respectively. Since
D. Tada et al. / Materials Science and Engineering C 27 (2007) 870–874
HSA and AL have been granted as pharmaceuticals or foods additive, respectively, the safety of resultant hydrogel is guaranteed. Since AL is anionic polysaccharide, the resultant hydrogel is expected to bind to both drugs with cationic charge and drugs having affinity to albumin. The present paper describes the preparation of hydrogels consisting of rHSA and AL and their in vitro evaluation as a drug carrier.
871
2.3. Swelling of hydrogels
2. Materials and methods
The hydrogel was left in distilled water until reaching to a constant size. Then, the weight of the hydrogel was measured after the moisture on the surface was removed with a filter paper. The hydrogel was also weighed after drying for a week in a desiccator. The swelling ratios (SRs) of hydrogels were calculated using SR = (Ws − Wd) / Wd, where Ws and Wd are the weight of the swollen and dried hydrogels, respectively.
2.1. Materials
2.4. Trypsin treatment of HSA-AL and EDA-AL hydrogels
Recombinant human serum albumin (rHSA) was kindly provided from Mitsubishi Pharma Corporation (Osaka). Sodium alginate 80–120 cP (AL), ethylenediamine (EDA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), and salicylic acid were purchased from Wako Pure Chemical Industries (Osaka). Sodium benzoate, o-anisic acid, and dibucaine hydrochloride were obtained from Sigma-Aldrich (St. Louis, MO, USA).
The hydrogels swollen in distilled water were immersed in Dulbecco's phosphate buffered saline (-) (DPBS) containing 0.25%(w/v) of trypsin at 37 °C for 2 weeks, changing trypsin solution everyday. The change in hydrogel appearance was recorded with photographs.
2.2. Preparation of rHSA-crosslinked alginate hydrogel (HSA-AL) AL was dissolved in 0.05 M 2-morpholinoethanesulfonic acid (MES) buffer (pH 5.6) and cooled to 4 °C on ice. EDC and NHS were added to AL solution to activate carboxylic acid groups of AL. After 20-minutes preactivation at 4 °C, rHSAwas added to the reaction mixture with gentle shaking. A combined weight of rHSA and AL was 600 mg and the volume of the mixture was adjusted to 12 ml with MES buffer. The amount of rHSA and AL actually used in hydrogel preparation is shown in Table 1. Then the reaction mixture was cast to perfluoro alkoxy fluoroplastics (PFA) dish (internal diameter: 50 mm) on which glass plate was placed to avoid drying and stood for 1 day at 4 °C. The resultant hydrogel was cut to a cylindrical shape by cork borer (diameter: 8.5 mm). Thus obtained hydrogel disk was washed in 50 g/l of Na2HPO4 solution and distilled water at 4 °C for 3 days, respectively. The hydrogel disk was stored in distilled water at 4 °C. The EDA-crosslinked AL hydrogels (EDA-AL) were prepared as a control in a similar manner to the HSA-AL. The compositions of the EDA-AL are also shown in Table 1.
Table 1 Preparation of hydrogels and their swelling ratios Hydrogel
HSA or EDA (mg)
HSA-AL (11:1) HSA-AL (5:1) HSA-AL (1:1) HSA-AL (1:5) EDA-AL (1:33) EDA-AL (1:1000)
550 500 300 100 18 0.6
a b c
AL (mg)
AL-COOH/HSA or EDA a (mol/mol)
Swelling ratio b (Ws/Wd) c
50 100 300 500 582 599
30 67 330 1670 10 320
44 ± 7 56 ± 7 81 ± 4 137 ± 14 41 ± 3 122 ± 18
The mol ratio of monosaccharide in AL to HSA or EDA. Each hydrogel came to equilibrium in distilled water. Ws and Wd are the weights of the swollen and dried hydrogels, respectively.
2.5. Adsorption of drugs to rHSA Twenty milliliters of rHSA aqueous solution (5 mg/ml) was placed in a seamless cellulose tubing (molecular cutoff of 12,000–16,000; Wako Pure Chemical Industries) and immersed in 380 ml of 0.53 mM drug solution at room temperature for 3 days, by which the drug concentration reached to a constant value. Then, the amount of the drug adsorbed to one rHSA molecule was calculated from the drug concentration in the outer solution. 2.6. Drug loading on a hydrogel Each drug was dissolved in distilled water to prepare 2 mM solution. Hydrogels swollen in distilled water were soaked in each drug solution at room temperature for 3 days. Then, the drug-loaded hydrogels were dried for 1 week in a desiccator. 2.7. Drug release from hydrogels The dried drug-loaded hydrogels (10 mg) were immersed in 50 ml of DPBS at room temperature. The medium was periodically replaced, and the amount of drug released into the medium was quantified by measuring the absorbance of the drug at 296 nm (salicylic acid and o-anisic acid in 1 M HCl), 224 nm (sodium benzoate), or 246 nm (dibucaine hydrochloride in 1 M HCl). After 2 weeks, the hydrogels were placed in DPBS at 60 °C for 24 h to complete the drug release. UV spectra were collected using a U-2010 spectrophotometer (Hitachi High Technologies, Tokyo). 3. Results and discussion 3.1. Hydrogel preparation Hydrogels consisting of rHSA and AL were prepared according to the procedure shown in Fig. 1. AL was firstly converted its activated ester by dehydrating condensation with NHS by the aid of water-soluble carbodiimide, EDC. Subsequently,
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Fig. 1. Preparation of HSA-AL hydrogel.
AL chains were crosslinked with HSA by the reaction of amino residues in HSA with activated ester groups in AL. Alginate hydrogels crosslinked with ethylene diamine (EDA) were similarly prepared as a control. The amount of HSA and AL used in hydrogels preparation and swelling ratio of resultant hydrogels are shown in Table 1. The increase of HSA or EDA feeding ratio to AL lowered the swelling ratio of a resultant hydrogel. HSA-AL hydrogels gradually swelled and reached to dissolution by the treatment with trypsin, while EDA-AL hydrogels retained their shape by the 2-weeks trypsin treatment (data not shown). Thus, HSA-AL hydrogel would gradually be cleared by the proteolysis of HSA in the body. 3.2. Drug loading HSA-AL hydrogel was loaded with salicylic acid, o-anisic acid, benzoic acid and dibucaine hydrochloride by immersing the hydrogel in each drug solution and subsequent drying. Table 2 shows the change in drug concentration before and after the hydrogel immersion into the drug solution. The data of o-anisic
acid were not shown in Table 2, since those were identical with those of benzoic acid. The concentration of dibucaine was heavily decreased by immersion of both HSA-and EDA-AL hydrogels and the decrease was more eminent for hydrogels of higher AL content, suggesting that the hydrogels adsorbed dibucaine through the ionic interaction of its positive charge with anionic AL. Among the three negatively charged drugs, only salicylic acid concentration slightly decreased when hydrogels of higher HSA content were used. This result suggests that salicylic acid was loaded on HSA-AL hydrogel by active adsorption of salicylic acid to HSA in addition to simple diffusion of the drug into a hydrogel. Then, these four drugs were briefly evaluated for the affinity to HSA by the method described in Materials and Methods. As shown in Fig. 2, the number of drugs bound to one HSA was determined to be 14 for salicylic acid, 5 for o-anisic acid, 3 for benzoic acid and 4 for dibucaine hydrochloride, supporting the results of drug loading. Although o-anisic acid and benzoic acid showed slight binding to rHSA, it might not be enough to change drug concentration upon drug loading.
Table 2 The drug concentration before and after the hydrogel immersion Hydrogel
HSA-AL (11:1) HSA-AL (5:1) HSA-AL (1:1) HSA-AL (1:5) EDA-AL (1:33) EDA-AL (1:1000)
Salicylic acid
Benzoic acid
Dibucaine
Before After (mM) (mM)
Before After (mM) (mM)
Before After (mM) (mM)
1.94 1.94 1.94 1.94 1.94
1.91 ± 0.01 1.92 ± 0.01 1.94 ± 0.00 1.94 ± 0.00 1.94 ± 0.00
1.92 1.92 1.92 1.94 1.94
1.91 1.91 1.91 1.91 1.91
1.90 ± 0.00 1.87 ± 0.01 1.75 ± 0.01 1.73 ± 0.02 1.50 ± 0.03
1.94
1.94 ± 0.00 1.94
1.94 ± 0.00 1.91
1.60 ± 0.01
1.94 ± 0.00 1.94 ± 0.00 1.94 ± 0.01 1.94 ± 0.00 1.94 ± 0.00
Fig. 2. Number of drug molecules adsorbed to one rHSA molecule. n = 3.
D. Tada et al. / Materials Science and Engineering C 27 (2007) 870–874 Table 3 Total amount of drug released from each hydrogel Hydrogel
Swelling Total release amount (μmol/10 mg gel) ratio a (Ws/Wd) b Salicylic o-Anisic Benzoic Dibucaine acid acid acid hydrochloride
HSA-AL (11:1) 44 ± 7 HSA-AL (5:1) 56 ± 7 HSA-AL (1:1) 81 ± 4 HSA-AL (1:5) 137 ± 14 EDA-AL (1:33) 41 ± 3 EDA-AL 122 ± 18 (1:1000) a b
2.99 ± 0.22 1.90 ± 0.12 0.75 ± 0.02 1.36 ± 0.03 0.37 ± 0.01 1.01 ± 0.03
0.78 ± 0.05 0.47 ± 0.05 0.63 ± 0.03 1.88 ± 0.04 0.48 ± 0.01 1.51 ± 0.01
0.66 ± 0.02 0.65 ± 0.01 0.80 ± 0.02 1.52 ± 0.08 0.45 ± 0.02 1.04 ± 0.08
3.5 ± 0.2 5.6 ± 0.2 16.1 ± 0.3 23.1 ± 2.8 29.0 ± 1.3 35.9 ± 0.3
Each hydrogel was left to swell in distilled water. Ws and Wd are the weights of the swollen and dried hydrogels, respectively.
3.3. Drug Release The drug release from each hydrogel was carried out by immersing the dried drug-loaded hydrogel in PBS at room temperature. Table 3 shows the total amount of drug released from a hydrogel, which might correspond to the amount of a drug loaded on a hydrogel, since further drug release was not observed when the hydrogel was incubated at 60 °C for 24 h after the drug release was ceased at room temperature. The amount of released dibucaine increased according to the increase of AL content in a hydrogel both for HSA-AL and EDA-AL hydrogels, reflecting the increase of ionic interaction between a cationic drug and anionic AL. When o-anisic acid and benzoic acid were loaded on HSA-AL hydrogels, the
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highest drug release was observed for HSA-AL (1:5) hydrogel, probably because of its highest swelling ratio. Although the release of these drugs decreased for HSA-AL (1:1) and (5:1) in a decreasing order of swelling ratio, the release slightly increased for HSA-AL (11:1) which had the lowest swelling ratio but the highest HSA content. This result is in good accordance with Fig. 2 indicating that both o-anisic acid and benzoic acid have slight binding ability to HSA. In contrast, salicylic acid was released most when loaded on HSA-AL (11:1) hydrogel and its release gradually decreased according to the decrease of HSA content up to 1:1 of HSA-AL ratio in spite of the increase of swelling ratio. These results coincide with those obtained in drug loading, indicating that salicylic acid was loaded on a hydrogel of higher HSA content mainly through binding to HSA, reflecting the high affinity of salicylic acid for HSA. Salicylic acid release was again raised when loaded on HSA-AL (1:5) hydrogel having highest swelling ratio, in which loading occur mainly through simple diffusion of the drug into a hydrogel along hydrogel swelling. Fig. 3 shows the time course of the release of each drug from HSA-AL hydrogels. As shown in Fig. 3a, salicylic acid release continued even after 24 h for HSA-AL (11:1) hydrogel and was completed within 4 h for HSA-AL (1:5) hydrogel. The release from HSA-AL (5:1) and (1:1) hydrogels were ceased around 10 h and 6 h, respectively. Thus, HSA-AL hydrogel of higher HSA content carried higher amount of salicylic acid and its release continued longer because of the binding to HSA. Benzoic acid and o-anisic acid release from HSA-AL (11:1), (5:1) and (1:1) hydrogels retained for about 5 h, while the release from HSA-AL (1:5) hydrogel
Fig. 3. Cumulative drug release from each hydrogels: (a) salicylic acid, (b) benzoic acid, (c) o-anisic acid and (d) dibucaine chloride. Symbols: closed circles, HSA-AL (11:1); closed triangles, HSA-AL (5:1); closed squares, HSA-AL (1:1), closed diamonds, HSA-AL (1:5), open circles, EDA-AL (1:33), and open diamonds, EDA-AL (1:1000). n = 3.
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ceased within 2 h (Fig. 3b and c), which was similar to that from EDA-AL hydrogels. These results also indicate that binding of benzoic acid and o-anisic acid to HSA took place to some extent in hydrogels of high HSA content. Release of dibucaine continued longer irrelevantly to HSA content of hydrogel, because of strong electrostatic interaction with AL (Fig. 3d). Thus, hydrogels consisting of recombinant HSA and AL have been prepared and evaluated as a drug carrier. Since HSA and AL have been granted as pharmaceuticals or foods additive, their safety is guaranteed. When HSA-AL hydrogel is administered to the body, the hydrogel would be solubilized by the proteolysis of HSA and resultant alginic acid would be excreted out of the body as demonstrated by uptake of alginic acid as foods. [12] From the present results showing sustained drug release for cationic drugs and the drugs having HSA affinity of HSA-AL hydrogel, HSA-AL hydrogel is expected to use as a safe sustained drug release carrier. Acknowledgement The authors are grateful to Mitsubishi Pharma Corporation for providing recombinant human serum albumin.
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