Influence of Substitutions and Enzymatic Hydrolysis by β-Mannanase

Jul 1, 2008 - Department of Fibre and Polymer Technology, School of Chemical Science and Engineering, Royal. Institute of Technology, Teknikringen 56-...
0 downloads 15 Views 429KB Size
Download PDF
2104

Biomacromolecules 2008, 9, 2104–2110

Articles Protein Release from Galactoglucomannan Hydrogels: Influence of Substitutions and Enzymatic Hydrolysis by β-Mannanase Alexandra Andersson Roos,† Ulrica Edlund,‡ John Sjo¨berg,‡ Ann-Christine Albertsson,‡ and Henrik Stålbrand*,† Department of Biochemistry, Lund University, Post Office Box 124, SE-221 00 Lund, Sweden, and Department of Fibre and Polymer Technology, School of Chemical Science and Engineering, Royal Institute of Technology, Teknikringen 56-58, SE-100 44 Stockholm, Sweden Received December 17, 2007; Revised Manuscript Received May 20, 2008

O-Acetyl-galactoglucomannan (AcGGM) is the major soft-wood hemicellulose. Structurally modified AcGGM and hydrogels of AcGGM were prepared. The degree of substitution (DS) of AcGGM was modified enzymatically with R-galactosidase, and chemically with an acrylate derivative, 2-hydroxyethylmethacrylate (HEMA). The hydrolysis of AcGGM with β-mannanase was shown to increase with decreasing DS. AcGGM hydrogels were prepared from chemically modified AcGGM with varying DS of HEMA. Bovine serum albumin (BSA) was encapsulated in hydrogels. A spontaneous burst release of BSA was decreased with increased DS of HEMA. The addition of β-mannanase significantly enhanced the BSA release from hydrogels with a DS of 0.36, reaching a maximum of 95% released BSA after eight hours compared to 60% without enzyme. Thus, both the pendant group composition and the enzyme action are valuable tools in the tailoring of hydrogel release profiles of potential interest for intestine drug delivery.

Introduction Polysaccharides are earning increased attention for food, packaging, agricultural, and biomedical applications where nontoxic, renewable, and degradable materials are needed. Polysaccharide hydrogels are suitable as biomaterials and have typically been utilized as bioadhesives, in tissue engineering, and in drug delivery.1 Colon-specific drug delivery2 is one of the target areas, where polysaccharides of interest are for example pectin, dextran, amylose, and cyclodextrin. Colonspecific drug release involves several possible approaches such as erosion and microbial activity.2 Hydrogels are particularly well suited for drug delivery applications due to their hydrophilicity, and soft permeable consistency. Furthermore, pH sensitive polysaccharide hydrogels have been designed with potential applications for intestinal delivery of protein drugs.3 A merit of natural polysaccharides is that each of them is susceptible to degradation by specific enzymes. Dextran hydrogels can be degraded by incubation with dextranase.4 In this article, we have focused on hydrogels of β-linked natural mannans. The backbone of mannans can be hydrolyzed by some bacteria of the colon,5 but the responsible enzymes are largely unknown. However, several β-mannanases from other microbes have been well characterized and are responsible for mannan endohydrolysis.6–8 The ultimate goal of our study was to synthesize hydrogels from O-acetyl-galactoglucomannan (AcGGM) with encapsulated bovine serum albumin (BSA), to * To whom correspondence should be addressed. E-mail: henrik. [email protected]. † Lund University. ‡ Royal Institute of Technology.

investigate the influence of substitutions and the feasibility of BSA-release mediated by the addition of β-mannanase to hydrolyze the hydrogel. AcGGM is the major soft-wood hemicellulose,9 which can be recovered10 from a paper pulp11 side stream. AcGGM has a O-acetylated β-(1,4)-linked glucomannan backbone with R-(1,6)galactosyl substitutions.12,13 The synthesis of AcGGM hydrogels involves the chemical introduction of acrylate substitutions and their cross-linking.14,15 Substitutions in general greatly influence the physicochemical behavior of polysaccharides,16,17 why we varied the degree of substitution (DS). The natural galactosyl substitutions of mannans influence the susceptibility to β-mannanase hydrolysis18 and possibly also the AcGGM properties, because mannans decorated with galactosyl side groups can be water solubilized, but unsubstituted mannans cannot.19,20 Therefore, we also varied the DS of galactosyl side groups by the use of R-galactosidase.21–23 Thus, here we target the influence of substitutions carried by AcGGM chains and hydrogels on properties, β-mannanase action, and release behavior. The motivation was to contribute to the understanding of AcGGM hydrogel properties and to the future possibility to design gels with desired release profiles for intestine drug delivery applications.

Material and Methods Materials. The following reagents for hydrogel preparation were used as received: N,N′-carbonyldiimidazole (g90%, Aldrich), 2-hydroxyethylmethacrylate (HEMA, >97%, Fluka), chloroform (99.8%, Labscan), triethylamine (>99.5%, Fluka), ethyl acetate (99.8%, Labscan), methanol (g98%, Labscan), dimethylsulfoxide (DMSO, g99.5%,

10.1021/bm701399m CCC: $40.75  2008 American Chemical Society Published on Web 07/01/2008

Protein Release from Galactoglucomannan Hydrogels Scheme 1. Procedure for Synthesis of AcGGM-Based Hydrogelsa

a

A schematic picture of an AcGGM fragment is shown in the box.

Riedel-de-Haen), ammonium peroxodisulphate (>98%, Fluka), and sodium pyrosulphite (g98%, Fluka). The used raw material was AcGGM originating from process water from thermomechanical pulping (TMP) of spruce provided by Stora Enso Kvarnsveden Mill AB, Sweden, purified by the diafiltration method, as previously described,10 and lyophilized. Purified hemicelluloses contained 77% AcGGM in a molar ratio of mannosyl/glucosyl/ galactosyl/acetyl of 1:0.3:0.2:0.4. The weight-average molecular weight was 14000, as determined by size-exclusion chromatography with AcGGM standards.24 Hydrogel Synthesis. AcGGM-based hydrogels were prepared through a three-step procedure14,15 (Scheme 1). In the first step, 2-[(1imidazolyl)formyloxy]ethyl methacrylate (HEMA-Im) was synthesized by mixing 2-hydroxyethylmethacrylate with N,N′-carbonyldiimidazole in anhydrous chloroform in room temperature for 1 h. The organic phase was extracted with water until the pH was neutral, dried over Na2SO4, and rotary evaporated under reduced pressure. The product was analyzed by nuclear magnetic resonance (NMR). 1H NMR (DMSOd6): δ 1.91 (s, 3 H, CH3), 4.48, 4.64 (m, 4 H, CH2-O), 5.59, 6.11 (s, 1 H each, vinyl C-H), and 7.05, 7.40, 8.11 (s, 1 H each, imidazole C-H). HEMA-Im was then covalently coupled to AcGGM by dissolving the reagents in DMSO with catalytic amounts of triethylamine. In a typical experiment, 1.5 g of AcGGM and 2 g of HEMA-Im were mixed and dissolved in DMSO. A total of 250 µL of triethylamine was added to initiate the reaction. The mixture was allowed to react at 50 °C for various times to achieve different degrees of substitution. HEMA-Im was reacted with AcGGM for 5 h to yield a DS of 0.1 (i.e., 10%) and for 17 h to give a DS of 0.36 (i.e., 36%). The product was isolated by precipitation in ethyl acetate or methanol followed by washing and centrifugation. The yellow powder was finally dried under vacuum. 1 H NMR (DMSO-d6): δ 1.88 (s, 3 H, CH3), 1.98-2.06 (s, 3 H, CH3CO, acetyl in the AcGGM chain), 3.2-5.1 (m, broad peaks from the AcGGM backbone), 4.30 (m, 4 H, CH2-O), 5.71, 6.06 (s, 1 H, each vinyl C-H). Hydrogels were finally prepared from AcGGM covalently modified with HEMA-Im using HEMA as a comonomer with the relative ratio: HEMA 60% (w/w) and modified AcGGM 40% (w/w). Cross-linking and simultaneous incorporation of 10% (w/w) BSA was performed in a water solution of HEMA, BSA, and modified AcGGM by adding catalytic amounts of water solutions of ammonium peroxodisulphate and sodium pyrosulfite, in order, and quickly transferring the solution to circular molds before gelation at room temperature. 1 H NMR Measurements. Synthesized structures were verified and the DS calculated by means of 1H NMR. Spectra were recorded on a

Biomacromolecules, Vol. 9, No. 8, 2008

2105

500 MHz Bruker DMX 500 spectrometer. Samples were dissolved in DMSO-d6 in 5 mm diameter sample tubes. The DSHEMA quantifies the relative amount of HEMA-Im groups coupled to AcGGM chains. DSHEMA was determined from 1H NMR spectra by relating the intensity of vinyl C-H peaks (on methacryloylated hydroxyl groups) to the intensity of acetyl CH3CO peaks (on anhydrohexose units). DSHEMA of hydrogels in this work ranged from 0.10 to 0.36. Enzymes. Purified R-galactosidase (AnGal27B, also designated R-gal III, AglB) from Aspergillus niger was used for the specific removal of galactosyl side groups from AcGGM. AnGal27B was purified as previously described21 and the activity of AnGal27B was assayed with p-nitrophenyl-R-D-galactopyranoside (Sigma, N-0877).25 The absorbance was measured at 400nm using p-nitrophenol as standards. The activity of the purified AnGal27B was determined to be 50.5 nkat mL-1 and the specific activity was determined to be 2665 nkat mg-1. A. niger β-mannanase (AnMan5A), purified as described,26 was used for the degradation of the β-linked mannan backbone of AcGGM and AcGGM based hydrogels. The activity of AnMan5A was assayed with locust bean gum using 3,5-dinitrosalicylic acid (DNS) to detect produced reducing sugars.27 The absorbance was measured at 540 nm with mannose as standard. The activity of the AnMan5A preparation was 55000 nkat mL-1 and the specific activity 5812 nkat mg-1. All enzyme activities are expressed in SI units (katals), and 1 nkat of activity is defined as the amount of enzyme that releases 1 nmol of p-nitrophenol per second or produces 1 nmol of reducing sugars per second under the conditions used. Protein Assay. Total protein determination of AnGal27B and AnMan5A mixtures was measured in microplates with the BCA Protein Assay Kit (Pierce, Rockford, IL) according to the manufactures instructions. The absorbance used was 562 nm. The BSA release from hydrogels was determined by direct measurement of the ultraviolet (UV) absorbance at 280 nm with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE). BSA was used as standards both for the BCA protein assay and the direct UV measurements. Analysis of Sugar Components. The monomer sugar composition of AcGGM was assayed by acid hydrolysis in 0.25 M sulfuric acid at 120 °C for 4 h as previously described.24,28,29 Monomeric sugars were analyzed before and after acid hydrolysis and the oligo- and polysaccharide content was calculated from the difference in monosaccharide concentration before and after hydrolysis. High performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) using an ED40 electrochemical detector (Dionex, Sunnyvale, CA, U.S.A.) was used to analyze the monomeric and oligomeric sugars. The chromatograph was equipped with a gradient pump (GP40, Dionex) and an autosampler (AS50, Dionex). The injection volume was 10 µL and the flow rate was 1 mL/min. To analyze monomeric sugars, Carbo Pac PA10 guard and analytical columns (Dionex) were used with degassed Millipore water as eluent. D-Mannose, D-glucose, D-galactose, D-xylose, and L-arabinose (Fluka Chemie AG) were used as standards. Carbo Pac PA100 guard and analytical columns (Dionex) were used with 100 mM sodium hydroxide as eluent to analyze oligomeric sugars. 1 D-Mannose (Fluka Chemie AG), mannobiose, mannotriose, and 6 -RD-galactosyl-mannotriose (Megazyme) were used as standards. Determination of Acetyl Content of AcGGM. AcGGM was dissolved in 1% NaOH to release the acetyl groups and treated overnight at room temperature.28 The released acetic acid was analyzed by high performance liquid chromatography (HPLC; GE Healthcare, Uppsala, Sweden) with an Aminex HPX-87H column (BIO-RAD, Hercules, CA, U.S.A.) at 65 °C with a refractive index detector (Erma-inc, Tokyo, Japan). The elution was performed with 0.005 M sulfuric acid, the flow rate was 0.6 mL/min and the injection volume was 100 µL. Acetic acid (Merck, Darmstadt, Germany) was used as standard. Substituted AcGGM. AcGGM with various substituents and hydrogels with AcGGM containing the varying substituents, were used

2106

Biomacromolecules, Vol. 9, No. 8, 2008

Roos et al.

Table 1. AcGGM and AcGGM-Based Hydrogels, with a Varying Degree of Substitution (DS)a degree of substitution (DS)b

free chains

hydrogels

DSGal

DSHEMA

designation

0.15 0.06 0.15 0.15 0.15 0.15 0.15 0.15 0.15

0 0 0.1 0.15 0.22 0.1 0.15 0.22 0.36

AcGGM 15:0 AcGGM 06:0 AcGGM 15:10 AcGGM 15:15 AcGGM 15:22 HG 15:10 HG 15:15 HG 15:22 HG 15:36

a All AcGGM substrates are referred to by the following designations. The DS of galactose and HEMA are based on total DS of the AcGGM. The DS of galactose was determined after acid hydrolysis and analysis of monosaccharides on HPAEC-PAD. The DS of HEMA was determined with NMR. b DSGal ) DS of galactose, DSHEMA ) DS of HEMA.

for degradation experiments with AnMan5A and are referred to by their designations listed in Table 1. Swelling Assessments. The dried gels were immersed in an excess of deionized water. At various times, the samples were withdrawn from the water phase and weighed. The swelling, Q, was then calculated from

Q ) (Ww-Wd) ⁄ Wd

(1)

where Wd is the weight of the dry gel prior to swelling and Ww is the weight in the swollen state. Galactosyl Removal with r-Galactosidase. Removal of the galactosyl side groups was performed by hydrolysing 2.5 mg/mL AcGGM in a test tube with 5 nkat AnGal27B/mg AcGGM in 100 mM sodium acetate buffer, pH 4.5. The hydrolysis mixture was incubated in a temperature of 40 °C on a shaker at 150 rpm. BSA (200 µg/mL) and sodium azide (400 µg/mL) were included in the incubation (BSA to stabilize the enzyme). Samples were withdrawn at different time intervals, terminated by boiling for 2 min and analyzed on HPAECPAD for galactose release. The total incubation was terminated after 96 h when 57% of the galactose residues had been removed. Total galactosyl content of the AcGGM was determined by acid hydrolysis in 0.25 M sulfuric acid at 120 °C for 4 h and the hydrolyzed monosaccharides were analyzed on HPAEC-PAD, as described above. β-Mannanase Hydrolysis of AcGGM- and AcGGM-Based Hydrogels. The β-mannanase AnMan5A26 from A. niger was used to study the enzymatic degradation of native and modified AcGGM and hydrogels made of chemically modified AcGGM. Hydrolysis of the AcGGM variants was performed in test tubes including 1 mg/mL AcGGM with the five different grades of substitution, listed in Table 1. The polysaccharides were incubated at 40 °C with 1 nkat AnMan5A/ mg AcGGM in 50 mM sodium acetate buffer, pH 4.5, BSA (100 µg/ mL), and sodium azide (200 µg/mL). Samples were withdrawn at different time intervals over a period of 48 h and terminated by the addition of DNS. The incubation mixture was vortexed before the samples were withdrawn and the total production of new reducing sugars was determined with addition of DNS, a boiling step for 10 min, and absorbance measurement at 540 nm. Hydrolysis of hydrogels prepared from chemically modified AcGGM, listed in Table 1, was performed at 40 °C with 10 mg dry hydrogel/ mL. The hydrogels were left to swell overnight in buffer before incubation with 1 nkat AnMan5A/mg dry hydrogel. The incubation buffer included 50 mM sodium acetate, pH 4.5, BSA (100 µg/mL), and sodium azide (200 µg/mL). Samples were withdrawn at different time intervals over a time period of 96 h and terminated by addition of DNS. The incubation mixture was vortexed before the samples were withdrawn. The total production of new reducing sugars was determined with DNS as described above. BSA Release from AcGGM-Based Hydrogels. The AnMan5Amediated release of BSA from hydrogels was studied in HG 15:10 gels (see Table 1) with BSA concentration of 10% (w/w). Hydrolyses with

1, 55, or 550 nkat AnMan5A/mg hydrogel were performed in test tubes at 40 °C with 10 mg dry hydrogel/mL. The incubation buffer included 50 mM sodium acetate, pH 4.5, and sodium azide (200 µg/mL). Everything, except the hydrogel, was mixed in the test tube and a sample was withdrawn to correlate for AnMan5A UV absorbance. A control, with no AnMan5A, was included in the experiment to detect the spontaneous BSA release from the hydrogel. The hydrogels were immersed into the buffer to start the hydrolysis. Samples were withdrawn at different time intervals over a time period of 48 h. The release of BSA was determined by measuring the UV absorbance. The released oligosaccharides were measured with DNS and HPAEC-PAD, as described above. The BSA release from hydrogels with higher DS (HG 15:36) and BSA concentration of 10 wt % (w/w) was investigated with an initial incubation for 24 h of the gel to remove spontaneous released BSA. After the initial incubation, hydrolysis with 55 nkat AnMan5A/ mg hydrogel was performed in test tubes at 40 °C with 10 mg dry hydrogel/ mL incubation mixture. The incubation mixture included 50 mM sodium acetate, pH 4.5, and sodium azide (200 µg/mL), and the incubation was performed as described above with samples withdrawn at different time intervals. The UV absorbance was measured on the Nanodrop and the released oligosaccharides were measured with DNS and HPAEC-PAD.

Results and Discussion In this work, we have studied the formation and release behavior of AcGGM-based hydrogels. Moreover, we have investigated β-mannanase catalyzed hydrolysis of the hydrogel AcGGM-backbone and its correlation to controlled release of an encapsulated model protein, BSA. Because hydrogel properties, β-mannanase action, and the release behavior is likely to be influenced by the AcGGM side groups, we prepared AcGGM and hydrogels with varying DS. The selective action of R-galactosidase was used to vary the DSGal, and DSHEMA was correlated to the time of the substitution reaction. The prepared free chains of AcGGM and the corresponding hydrogels are listed in Table 1. Enzymatic Hydrolysis of Modified AcGGM. Thus, the side group composition of AcGGM chains was purposely altered using two different strategies: enzymatic action and synthetic coupling of acrylate groups. In the first case, R-galactosidase was used: an enzyme activity previously used to alter the structure of galactomannan gums.30 By incubation with R-galactosidase AnGal27B in acidic media, a cleavage of galactosyl side groups from the AcGGM backbone was afforded. The initial composition of spruce AcGGM recovered from TMP pulping had a molar ratio of mannosyl/ glucosyl/galactosyl/acetyl of 1:0.3:0.2:0.4. Thus, the degree of galactosyl substitution of the glucomannan backbone, DSGal, was 0.15. After structural modification with AnGal27B for 96 h, the DSGal of the AcGGM was 0.06. In the second case, AcGGM chains were modified synthetically. HEMA-Im was synthesized in a separate step and then covalently attached to the AcGGM backbone by an exchange reaction with its hydroxyl groups producing a polysaccharide chain with pendant HEMA groups. The number of HEMA groups attached, quantified as DSHEMA, is directly controlled through the reaction time. Chemical modifications were prepared from AcGGM with a DSGal of 0.15 to form AcGGM with DSHEMA of 0.1, 0.15, and 0.22. Five different AcGGM variants were thus prepared: DSGal ) 0.15 and 0.06, respectively, and DSHEMA ) 0.1, 0.15, and 0.22. These five formulations were hydrolyzed with AnMan5A (1 nkat/mg AcGGM) over a time period of 48 h to explore the

Protein Release from Galactoglucomannan Hydrogels

Biomacromolecules, Vol. 9, No. 8, 2008

2107

Figure 2. The swelling behavior of AcGGM-based hydrogels (HG 15: 36 solid line and HG 15:10 hatched line). See Table 1 for hydrogel designation. Table 2. Maximum Hydrolysis after 24 or 48 h and Hydrolysis Ratesa substrate AcGGM AcGGM AcGGM AcGGM AcGGM

Figure 1. Hydrolysis with AnMan5A of (A) AcGGM 15:0 (solid line) and AcGGM 06:0 (dashed line), and (B) AcGGM 15:0 (solid line), AcGGM 15:10 (dashed line), AcGGM 15:15 (dotted line), and AcGGM 15:22 (dash/dotted line). The same AcGGM 15:0 hydrolysis data was included in both graphs for comparison of the different DS. The concentration of produced reducing sugars is shown.

initial hydrolysis rate and the maximum hydrolysis. The initial hydrolysis rate was determined after 40 min as mole produced reducing sugars s-1 mole enzyme-1 added. As seen in Figure 1, the hydrolysis progressed similarly for all samples in the sense that they all displayed an initial rapid increase of the production of new reducing sugars created by AnMan5A. For all samples, a retardation is apparent after about 1-1.5 h of hydrolysis (inset in Figure 1). A closer look revealed that even though the trend was similar, the initial hydrolysis rate did differ from one AcGGM variant to another and that the DS was a significant factor in this respect. The original AcGGM starting material (AcGGM 15:0) yielded an intial hydrolysis rate of 59 mol s-1 mole-1, which increased to 66 mol s-1 mole-1 up on partial removal of galactosyl substitution (AcGGM 06:0). The trend was the same with acrylate substitutions. When AcGGM was used as a starting material, a higher DSHEMA decreased the enzymatic hydrolysis rate: AcGGM 15:10, 38 mol s-1 mole-1 and AcGGM 15:15, 32 mol s-1 mole-1. The results are given in Table 2. The maximum hydrolysis achieved was assessed by analyzing the total amount of produced reducing sugars after 48 h (Table 2). The results show that both the side group type and content are important factors governing the hydrolysis yield. Following the same trend as the intial hydrolysis rate, the maximum yield decreased with higher DS for both acrylate and galactosyl

06:0 15:0 15:10 15:15 15:22

hydrolysis rate at 0-40 minb

maximum hydrolysisc (mM produced reducing sugars)

66.9 58.9 37.8 31.6 25.5

2.3(24h) 1.8(24h) 1.6(24h) 1.4(48h) 1.2(48h)

a At 40 min of unmodified, enzymatically, and chemically modified AcGGM with hydrolysis of 1 nkat AnMan5A/mg AcGGM. b The hydrolysis rate after 40 min was calculated as mole produced reducing sugars s-1 mol-1 AnMan5A. c Maximum hydrolysis is calculated as mM produced reducing sugars from 1 mg/mL AcGGM at the highest observed concentration, which were 24 or 48 h.

substitutions, and the two highest substituted chains took a longer time (48 h) to reach maximum yield compared to the others (24 h; Table 2). The observed behavior may be explained in terms of backbone obstruction. The hydrolysis with AnMan5A has earlier been shown to be hindered by side group substitution of acetyl groups31,32 and galactosyl groups18,33 of mannans and galactomannans. HEMA, when covalently linked to AcGGM, is a side group that in a similar manner may sterically hinder the AnMan5A action on the mannan backbone. It is, therefore, reasonable to assume that more side groups carried by the glucomannan backbone will give slower hydrolysis rate and less amounts of sites on the backbone to be hydrolyzed, thus resulting in a lower maximum hydrolysis. Hydrogels from Modified AcGGM: Swelling. Hydrogels were prepared from AcGGM chains modified with HEMA side groups, as described above (see designations in Table 1). By deliberate variation of the reaction time, formulations with a high and a low DSHEMA, respectively, were produced: DSHEMA ) 0.36 and 0.1. The modified AcGGM chains were then crosslinked in the presence of 60% (w/w) HEMA to form a covalently linked interconnected structure. The resulting materials were swollen in an excess of deionized water (Figure 2). Showing a significant swelling capacity, these materials may correctly be termed hydrogels. Hydrogels with the lower DSHEMA (HG 15: 10) had a higher swelling capacity than hydrogels with higher DSHEMA (HG 15:36), in accordance with our previous results14,15 on AcGGM hydrogels. This is due to the higher degree of crosslinking of the latter stemming from the higher number acrylate groups attached to the AcGGM chain. Each acrylate group

2108

Biomacromolecules, Vol. 9, No. 8, 2008

Roos et al.

Figure 3. Hydrolysis with AnMan5A of chemically (HEMA) modified AcGGM hydrogel, HG 15:10 (solid line), HG 15:15 (dashed line), and HG 15:22 (dotted line). The concentration of reducing sugars produced is shown.

Figure 4. Spontaneous (solid line) and AnMan5A catalyzed (1 nkat/ mg hydrogel, dashed line; 55 nkat/mg hydrogel, dotted line; 550 nkat/ mg hydrogel, dashed/dotted line) BSA release from HG 15:10 hydrogels.

Table 3. Maximum hydrolysis after 96 h and Hydrolysis Rates after 24 h of Chemically Modified AcGGM Hydrogelsa

important. Accessibility of the enzyme to the AcGGM backbone is still believed to be a significant factor because the hydrogel with the highest DSHEMA produced the slowest hydrolysis with the lowest maximum yield. However, other factors need to be considered to explain why the gel with the lowest DS did not provide the fastest degradation. Intramolecular segment association may give rise to the described behavior. Unsubstituted stretches of mannan in, for example, galactomannans have been reported to interact as to form partly associated chains.34–37 If a similar effect arises in these hydrogel formulations, the associated regions would in practice act as physical cross-links, producing a more tightly linked network than the covalent linkages alone. As this effect would be most pronounced in the hydrogel with the longest unsubstituted stretches of the glucomannan backbone, the hydrogel with the lowest DS would constitute a greater obstacle to enzymatic cleavage than a gel with a somewhat higher amount of substituted groups, consistent with the AcGGM 15:10 gel being more slowly hydrolyzed than the AcGGM 15:15 gel. With even higher DS, the amount of covalent linkages is apparently becoming the overriding effect, and the sterical hindrance again leads to a slower hydrolysis. BSA Release from Hydrolyzed Hydrogels. Hydrogels prepared from AcGGM chains with a low and a high DSHEMA, respectively (HG 15:10 and HG 15:36), were chosen to demonstrate the controlled release capacity of AcGGM hydrogels and the role of enzymatic action on the release rate and control. A total of 10% (w/w) of BSA was incorporated into the hydrogels and the release in aqueous media was pursued with and without the presence of the AcGGM hydrolysing enzyme β-mannanase AnMan5A. Starting with the HG 15:10 formulation, three different enzyme loadings of β-mannanase (1 nkat, 55 nkat and 550 nkat/mg hydrogel) were examined and compared with the release pattern displayed when incubating the hydrogel in the absence of enzyme. Samples were withdrawn at different time intervals during 48 h. The amount of release BSA was monitored with UV absorbance at various time intervals. As shown in Figure 4, there is an initial burst of BSA release from all hydrogels, also from the gel with no AnMan5A addition. However, with the addition of AnMan5A, the release rate of BSA is faster than for the hydrogel with no added AnMan5A. The effect of AnMan5A can be seen more clearly after a few hours when the initial burst of BSA release has leveled out. The maximum release of BSA is higher for hydrogels treated

substrate

hydrolysis rate at 0-24 hb

maximum hydrolysis after 96 hc (mM produced reducing sugars)

HG 15:10 HG 15:15 HG 15:22

0.889 1.16 0.775

7.17 8.99 6.29

a Hydrolysis was performed with 1 nkat AnMan5A/mg hydrogel. b The hydrolysis rate after 24 h was calculated as moles produced reducing sugars s-1 mol-1 AnMan5A. c Maximum hydrolysis is calculated as mM produced reducing sugars from 10 mg/mL hydrogel at 96 h.

provides a vinyl functionality that reacts by radical polymerization in the cross-linking step. Thus, a higher DSHEMA give rise to a tighter network and the capacity to hold water is reduced. Hydrogels from Modified AcGGM: Enzymatic Hydrolysis. To explore the enzymatic hydrolysis, hydrogels were prepared as described in the previous paragraph. HEMA side groups were attached to the AcGGM backbone to three different extents, DSHEMA ) 0.1 (AcGGM 15:10), DS ) 0.15 (AcGGM 15:15), and DS ) 0.22 (AcGGM 15:22). Gels were formed by cross-linking in the presence of additional HEMA so that total amount of modified AcGGM was 40%. Hydrolysis was afforded by treating these hydrogels with 1 nkat β-mannanase/mg dry hydrogel. Figure 3 and Table 3 shows the AnMan5A hydrolysis in terms of rate and maximum degradation achieved for hydrogels containing the various substituted AcGGM chains. The data clearly show that the hydrolysis behavior of crosslinked AcGGM chains is not as clear-cut as in the case of free chains. For free chains, a higher DSHEMA inevitably led to a slower rate and a lower maximum hydrolysis, as discussed above (Figure 1 and Table 2). For hydrogels, however, the highest hydrolysis rate was obtained with a DSHEMA of 0.15 (1.2 moles produced reducing sugars s-1 mole-1 AnMan5A), with the second highest hydrolysis rate at DSHEMA 0.1 (0.90 moles s-1 mole-1) (Figure 3, Table 3). The slowest hydrolysis rate was detected for DSHEMA 0.22 (0.78 moles s-1 mol-1). The same trend arose for maximum hydrolysis, where the highest maximum hydrolysis was obtained with DSHEMA of 0.15 (9.0 mM) with the second highest hydrolysis obtained with DSHEMA of 0.1 (7.2 mM). The lowest maximum hydrolysis was obtained with DSHEMA of 0.22 (6.3 mM). The results indicate that, with the hydrogels, effects other than just steric hindrance by the acrylate side groups are

Protein Release from Galactoglucomannan Hydrogels

Biomacromolecules, Vol. 9, No. 8, 2008

2109

Conclusions

Figure 5. Spontaneous (solid line) and AnMan5A catalyzed BSA release from HG 15:36 (dashed line). AnMan5A (55 nkat/mg hydrogel) was added to the incubation after 24 h.

with more AnMan5A. The maximum release of BSA after 48 h is 7.8% more with 1 nkat/mg, 16% more with 55 nkat/mg, and 40% more with 550 nkat/mg hydrogel than the release from the control hydrogel without AnMan5A addition. A higher enzyme loading thus yields a higher total release, also reflected by a higher hydrolysis, judged from the production of hydrolysis products. Analysis with HPAEC-PAD revealed that predominantly smaller oligosaccharides than a degree of polymerization (DP) of four had been released (data not shown). Calculations of mono- and oligosaccharides with DP e 3 after 48 h of hydrolysis showed that 0.45 mM mono- and oligosaccharides for 1 nkat, 1.1 mM for 55 nkat, and 2.8 mM for 550 nkat AnMan5A had been released. Total concentrations of released reducing sugars determined with the DNS assay were 2.8 mM for 1 nkat and 4.3 mM for 55 nkat and >7 mM for 550 nkat. AnMan5A-mediated BSA release from hydrogels with higher DS (HG 15:36) was performed. First, the hydrogel was incubated in buffer without AnMan5A for 24 h to wash out spontaneously released BSA. The released BSA was 0.55 mg/ mL (55%; Figure 5). Then, after 24 h, AnMan5A was added and incubated along with a control without enzyme addition. With the control only minor amounts of BSA was released. Upon AnMan5A addition however, a notably increase of BSA release was observed resulting in a concentration of 0.95 mg/mL (95%) 8 h after enzyme addition and thereafter only minor quantities. The results altogether show that incorporation of BSA in AcGGM hydrogels is a viable route of achieving sustained release. Previous work have demonstrated the potential of GGM-based hydrogel formulations in the release of incorporated molecules.38,39 As shown here, the process is mediated by enzyme catalysis, providing an effective steering parameter to achieve a preferable release time. In summary, hydrogels from AcGGM are useful to achieve a prolonged release of a model protein. The spontaneous release of the target molecule incorporated into the hydrogel is decreased with an increase of the acrylate content of modified AcGGM. β-Mannanase-mediated backbone hydrolysis enhances the erosion of the gel and increases the release of the target molecule. From Figure 5 it can be concluded that after a wash step of 24 h, BSA release from AcGGM-based hydrogels can in essence be totally controlled by β-mannanase hydrolysis of the AcGGM backbone.

Incubation with the R-galactosidase AnGal27B and the synthetic coupling of acrylate pendant groups were independently shown to be viable pathways for modifying the polysaccharide structure of AcGGM, and the extent of structural substitution is controllable via the reaction time. The resulting side group structure and content of native and modified AcGGM chains had in turn a marked effect when these chains were successfully degraded by β-mannanase-mediated hydrolysis. Increased side group substitution of modified AcGGM decreased the hydrolysis rate and maximum hydrolysis, indicating steric hindrance of the enzyme by the acrylate side group. Hydrogels were prepared from AcGGM substituted with various amounts of 2-hydroxyethylmethacrylate groups and loaded with BSA. Two parameters were found that influence the release of BSA from the hydrogels in water; the degree of substitution of HEMA and the presence of β-mannanase AnMan5A. Less BSA was released spontaneously with an increase of HEMA substitutions on the glucomannan backbone from 0.1 to 0.36, while the addition of β-mannanase AnMan5A increased the BSA release. Aided by the enzymatic hydrolysis of AcGGM, the hydrogel with DSHEMA of 0.36 released almost all remaining BSA from the hydrogel within 8 h after addition. These results provide valuable insights into further developments of AcGGM-based hydrogels for the application of drug delivery. Acknowledgment. The Swedish Agency for Innovation Systems (VINNOVA) is gratefully acknowledged for financial support (Hemigels project 2004-01558). The Swedish Research Council (VR) is gratefully thanked for financial support to H.S.

References and Notes (1) Chen, J.; Jo, S.; Park, K. Polysaccharide hydrogels for protein drug delivery. Carbohydr. Polym. 1995, 28 (1), 69–76. (2) Sinha, V. R.; Kumria, R. Polysaccharides in colon-specific drug delivery. Int. J. Pharm. 2001, 224 (1-2), 19–38. (3) George, M.; Abraham, T. E. pH sensitive alginate-guar gum hydrogel for the controlled delivery of protein drugs. Int. J. Pharm. 2007, 335, 123–129. (4) Franssen, O.; Stenekes, R. J. H.; Hennink, W. E. Controlled release of a model protein from enzymatically degrading dextran microspheres. J. Controlled Release 1999, 59 (2), 219–228. (5) Macfarlane, G. T.; Hay, S.; Macfarlane, S.; Gibson, G. R. Effect of different carbohydrates on growth, polysaccharidase and glycosidase production by Bacteroides oVatus, in batch and continuous culture. J. Appl. Bacteriol. 1990, 68, 179–187. (6) Hilge, M.; Gloor, S. M.; Rypniewski, W.; Sauer, O.; Heightman, T. D.; Zimmerman, W.; Winterhalter, K.; Piontek, K. High-resolution native and complex structures of thermostable β-mannanase from Thermomonospora fusca-substrate specificity in glycosyl hydrolase family 5. Structure 1998, 6, 1433–1444. (7) Hogg, D.; Woo, E.-J.; Bolam, D. N.; McKie, D. A.; Gilbert, H. J.; Pickersgil, R. W. Crystal structure of mannanase 26A from Pseudomonas cellulosa and analysis of residues involved in substrate binding. J. Biol. Chem. 2001, 276, 31186–31192. (8) Le Nours, J.; Anderson, L.; Stoll, D.; Stålbrand, H.; Leggio, L. L. The structure and characterisation of a modular endo-β-1,4-mannanase from Cellulomonas fimi. Biochemistry 2005, 44, 12700–12708. (9) Sjo¨stro¨m, E. Wood Chemistry Fundamentals and Applications, 2nd ed.; Academic Press Inc.: London, 1993; pp 63-70. (10) Andersson, A.; Persson, T.; Zacchi, G.; Stålbrand, H.; Jo¨nsson, A.-S. Comparison of diafiltration and size-exclusion chromatography to recover hemicelluloses from process water from thermomechanical pulping of spruce. Appl. Biochem. Biotechnol. 2007, 136-140, 971– 984. (11) Varhimo, A.; Tuovinen, O. Raw materials. In Mechanical Pulping; Sundholm, J., Ed.; Fapet, Cop: Helsinki, 1999; Vol. 5, pp 66-104. (12) Timell, T. E. Recent progress in the chemistry of wood hemicellulose. Wood Sci. Technol. 1967, 1, 45–70.

2110

Biomacromolecules, Vol. 9, No. 8, 2008

(13) Lindberg, B.; Rosell, K.-G.; Svensson, S. Position of the O-acetyl groups in pine glucomannan. SVensk Papperstidning 1973, 76, 383– 384. (14) So¨derqvist Lindblad, M.; Ranucci, E.; Albertsson, A.-C. Biodegradable polymers from renewable sources. New hemicellulose-based hydrogels. Macromol. Rapid Commun. 2001, 22 (12), 962–967. (15) Lindblad, M. S.; Albertsson, A.-C.; Ranucci, E.; Laus, M.; Giani, E. Biodegradable polymers from renewable sources: Rheological characterization of hemicellulose-based hydrogels. Biomacromolecules 2005, 6 (2), 684–690. (16) Balser, K.; Hoppe, L.; Eicher, T.; Wandel, M.; Astheimer, H.-J.; Steinmeier, H. Cellulose Esters. Ullmann’s Encyclopedia of Industrial Chemistry, 7th ed.; Wiley-VCH: Weinheim, Germany, 2004; Electronic release. (17) Brandt, L. Cellulose Ethers. In Ullman’s Encyclopedia of Industrial Chemistry; Campbell, F. T.; Pfefferkorn, R.; Rounsaville, J. F., Eds.; VCH Verlagsgesellschaft: Weinheim, Germany, 1986; Vol. A5, p 461. (18) McCleary, B. V.; Matheson, N. K. Action patterns and substratebinding requirements of beta-D-mannanase with mannosaccharides and mannan-type polysaccharides. Carbohydr. Res. 1983, 119, 191– 219. (19) Meier, H. On the structure of cell walls and cell wall mannans from ivory nuts and from dates. Biochim. Biophys. Acta 1958, 28, 229– 240. (20) Dea, I. C. M.; Morrison, A. Chemistry and interactions of seed galactomannans. AdV. Carbohydr. Chem. Biochem. 1975, 31, 241– 312. (21) Ademark, P.; Larsson, M.; Tjerneld, F.; Stålbrand, H. Multiple R-galactosidase from Aspergillus niger: Purification, characterization, and substrate specificities. Enzyme Microb. Technol. 2001, 29, 441– 448. (22) de Vries, R. P.; van den Broeck, H. C.; Dekkers, E.; Manzanares, P.; de Graaff, L. H.; Visser, J. Differential expression of three alphagalactosidase genes and a single β-galactosidase gene from Aspergillus niger. Appl. EnViron. Microbiol. 1999, 65 (6), 2453–60. (23) Manzanares, P.; de Graaff, L. H.; Visser, J. Characterization of galactosidases from Aspergillus niger: Purification of a novel R-galactosidase activity. Enzyme Microb. Technol. 1998, 22 (5), 383–90. (24) Lundqvist, J.; Jacobs, A.; Palm, M.; Zacchi, G.; Dahlman, O.; Stalbrand, H. Characterization of galactoglucomannan extracted from spruce (Picea abies) by heat-fractionation at different conditions. Carbohydr. Polym. 2003, 51 (2), 203–211. (25) Ra¨tto¨, M.; Poutanen, K. Production of mannan-degrading enzymes. Biotechnol. Lett. 1988, 10 (9), 661–664. (26) Ademark, P.; Varga, A.; Medve, J.; Harjunpa¨a¨, V.; Drakenberg, T.; Tjerneld, F.; Stålbrand, H. Softwood hemicellulose-degrading enzymes

Roos et al.

(27) (28) (29)

(30) (31)

(32)

(33) (34) (35)

(36)

(37) (38) (39)

from Aspergillus niger: Purification and properties of a β-mannanase. J. Biotechnol. 1998, 63 (3), 199–210. Stålbrand, H.; Siika-aho, M.; Tenkanen, M.; Viikari, L. Purification and characterization of two β-mannanases from Trichoderma reesei. J. Biotechnol. 1993, 29, 229–242. Lai, Y.-Z. Chemical Degradation. In Wood and Cellulosic Chemistry, 1st ed.; Shiraishi N., Ed.; Dekker Marcel: New York and Basel, 1991; Vol. 10, pp 455-522. Wright, J. D.; Power, A. J. Comperative technical evaluation of acid hydrolysis processes for conversion of cellulose to alcohol. In Energy from biomass and wastes x; Klass, E. D., Ed.; Elsevier Applied Science Publisher: London, 1987; pp 949-971. Burke, M. D.; Park, J. O.; Srinivasarao, M.; Khan, S. A. A novel enzymatic technique for limiting drug mobility in a hydrogel matrix. J. Controlled Release 2005, 104 (1), 141–153. Oosterveld, A.; Coenen, G. J.; Vermeulen, N. C. B.; Voragen, A. G. J.; Schols, H. A. Structural features of acetylated galactomannans from green Coffea arabica beans. Carbohydr. Polym. 2004, 58 (4), 427– 434. Puls, J.; Schuseil, J. Chemistry of hemicelluloses: relationship between hemicellulose structure and enzymes required for hydrolysis. In Hemicellulose and Hemicellulases; Coughlan, M. P., Hazlewood, G. P., Eds.; Portland Press Res. Monogr.: London, 1993; Vol. 4, pp 1-27. Ganter, J. L. M. S.; Sabbi, J. C.; Reed, W. F. Real-time monitoring of enzymatic hydrolysis of galactomannans. Biopolymers 2001, 59 (4), 226–242. Morris, E. R.; Cutler, A. N.; Ross-Murphy, S. B.; Rees, D. A.; Price, J. Concentration and shear rate dependence of viscosity in random coil polysaccharide solutions. Carbohydr. Polym. 1981, 1 (1), 5–21. Dea, I. C. M.; Morris, E. R.; Rees, D. A.; Welsh, E. J.; Barnes, H. A.; Price, J. Associations of like and unlike polysaccharides: Mechanism and specificity in galactomannans, interacting bacterial polysaccharides, and related systems. Carbohydr. Res. 1977, 57, 249–272. Dea, I. C. M.; Clark, A. H.; McCleary, B. V. Effect of the molecular fine structure of galactomannans on their interaction propertiessThe role of unsubstituted sides. Food Hydrocolloids 1986, 1 (2), 129– 140. Goycoolea, F. M.; Morris, E. R.; Gidley, M. J. Viscosity of galactomannans at alkaline and neutral pH: Evidence of “hyperentanglement” in solution. Carbohydr. Polym. 1995, 27 (1), 69–71. Voepel, J.; Sjo¨berg, J.; Albertsson, A.-C.; Hultin, U.-K.; Gasslander, U. Drug diffusion in neutral and ionic hydrogels assembled from acetylated galactoglucomannan. J. Appl. Polym. Sci. 2008, submitted. Edlund, U.; Albertsson, A.-C. A microspheric system: Hemicellulosebased hydrogels. J. Bioact. Compat. Polym. 2008, 23, 171–186.

BM701399M