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Biomaterials 25 (2004) 24612466

Controlled degradation of hydrogels using multi-functional cross-linking molecules


Kuen Yong Leea, Kamal H. Bouhadira,b, David J. Mooneya,*
a

Departments of Biologic & Materials Sciences and Chemical Engineering, University of Michigan, 3074 H.H. Dow Building, 2300 Hayward Street, Ann Arbor, MI 48109-2136, USA b Department of Chemistry, American University of Beirut, Beirut 110236, Lebanon Received 31 March 2003; accepted 4 September 2003

Abstract Hydrogels, chemically cross-linked or physically entangled, have found a number of applications as novel delivery vehicles of drugs and cells. However, the narrow ranges of degradation rates and mechanical strength currently available from many hydrogels limits their applications. We have hypothesized that utilization of multi-functional cross-linking molecules to form hydrogels could provide a wider range and tighter control over the degradation rates and mechanical stiffness of gels than bi-functional cross-linking molecules. To address the possibility, we isolated a-l-guluronate residues of sodium alginate, and oxidized them to prepare poly(aldehyde guluronate) (PAG). Hydrogels were formed with either poly(acrylamide-co-hydrazide) (PAH) as a multi-functional cross-linking molecule or adipic acid dihydrazide (AAD) as a bi-functional cross-linking molecule. The initial properties and degradation behavior of both PAG gel types were monitored. PAG/PAH hydrogels showed higher mechanical stiffness before degradation and degraded more slowly than PAG/AAD gels, at the same concentration of cross-linking functional groups. The enhanced mechanical stiffness and prolonged degradation behavior could be attributed to the multiple attachment points of PAH in the gel at the same concentration of functional groups. This approach to regulating gel properties with multifunctional cross-linking molecules could be broadly used in hydrogels. r 2003 Elsevier Ltd. All rights reserved.
Keywords: Degradation; Cross-linking; Alginate; Drug delivery; Tissue engineering

1. Introduction Hydrogels have found numerous applications in the delivery of drugs and cells [1,2], as they can be used as injectables to avoid radical surgery and reduce patients pain. Investigators have focused on controlling mechanical properties and degradation behavior of hydrogels, as well as on enhancing their biological interactions with body components, in order to design and tailor appropriate vehicles for drug delivery and tissue engineering applications [35]. For example, the mechanical stiffness of hydrogels is often desired to be similar to the range of tissues in the body, and degradation rates of hydrogels may be designed to coincide with the rate of new tissue formation in tissue
*Corresponding author. Tel.: +1-734-763-4816; fax: +1-734-7630459. E-mail address: mooneyd@umich.edu (D.J. Mooney). 0142-9612/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2003.09.030

engineering approaches [6]. However, the desired mechanical strength and degradation rate may vary greatly for different applications [7]. Controlling degradation behavior has been one of critical issues in general biomaterials research, and has been widely investigated to date [810]. In general, biomaterials need to be cleared from the body once they complete their roles in the body, and degradable materials could be ideal for this purpose. Two approaches are typically used to obtain degradable hydrogels. In the rst, gelling polymers are designed such that their backbone is degradable by hydrolysis and/or enzymatic action. The second approach involves introduction of degradable cross-linking points to systems that are comprised of non-degradable polymer chains. Many polymers, including aliphatic polyesters and collagen, have been identied to undergo main chain scission by hydrolysis and enzymatic action, respectively [11,12]. Covalent or photo-cross-linking

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of modied poly(ethylene glycol)s with degradable cross-links would t into the second approach, as poly(ethylene glycol) is degradable neither by hydrolysis nor enzymes [13,14]. Alginate, a typical biomaterial used to form hydrogels, has a blockwise structure composed of a-lguluronate and b-d-mannuronate, and their relative amounts in alginate signicantly inuences the rheological properties of the alginate solution as well as the mechanical properties of the resultant gels [15]. Alginate forms gels easily in the presence of divalent cations under mild conditions [16], and its gels have been extensively utilized in drug delivery and cell encapsulation applications [1719]. However, ionically crosslinked gels exhibit uncontrolled degradation behavior due to the diffusion of divalent ions from the gels in physiological conditions [20]. We have previously demonstrated that certain ranges of mechanical strength and degradation rates of alginate-derived gels can be controlled by using covalent cross-linking with bifunctional molecules [21]. However, the mechanical stiffness of the gels is often relatively low and the gels often degrade rapidly in vitro unless excess amounts of cross-linking molecules are used. We hypothesized that multi-functional cross-linking molecules, which provide multiple attachments to hydrogels, could result in stronger gels and more tightly controlled degradation behavior than those cross-linked with bi-functional cross-linking molecules. In this context, we synthesized a multi-functional molecule to cross-link alginate-derived polysaccharides, and compared their mechanical properties and degradation behavior with those cross-linked with bi-functional molecules at the same concentration of functional groups. In brief, polyguluronate (PG), composed of al-guluronate residues, was isolated from alginate by acid hydrolysis, oxidized, and covalently cross-linked with either adipic dihydrazide (bi-functional crosslinker) or poly(acrylamide-co-hydrazide) (PAH; multifunctional cross-linker) to form hydrogels. Degradation behavior of these gels were monitored by measuring the weight loss, elastic modulus, and cross-linking density of the gels as a function of degradation time. 2. Materials and methods 2.1. Materials Sodium alginate with high guluronate content (Protanal LF 20/40) was purchased from Pronova (Drammen, Norway). Polyacrylamide and hydrazine were purchased from Sigma (St. Louis, MO) and used without further purication. Dulbeccos modied Eagles medium (DMEM) was purchased from Life Technologies (Grand Island, NY). All chemical reagents were analytical grade and used as received.

2.2. Synthesis of PAH Polyacrylamide (50 wt%, molecular weight B10,000, 50 ml) was mixed with aqueous hydrazine solution (35 wt%, 100 ml), and the mixture was reuxed for 3 h at room temperature. The resultant solution was evaporated in a vacuum using a rotary evaporator, and then dissolved in distilled water (200 ml). The solution was dialyzed against distilled water for 3 days (MWCO 1000, Spectra/Pors), concentrated using an Amicons concentrator under reduced pressure, and lyophilized. The degree of substitution of hydrazide groups to polyacrylamide was spectrophotometrically determined at 334 nm using trinitrobenzenesulfonic acid (TNBS). 2.3. Formation of PAG hydrogels PAG was prepared according to the previously reported method [21,22]. In brief, PG was isolated from sodium alginate by acid hydrolysis and collected at pH 2.85. PG was then oxidized with 0.25 m sodium periodate solution at room temperature. The ratio between guluronate unit and periodate was 1:1, and an equimolar amount of ethylene glycol to guluronate units was added after 19 h to stop the oxidation. The resultant solution was dialyzed against distilled water (MWCO 1000, Spectra/Pors) for 3 days, concentrated under reduced pressure, and lyophilized. A PAG solution was mixed with different amounts of either adipic acid dihydrazide (AAD) or PAH, placed into 48-well tissue culture plates, and incubated at room temperature for 4 h to form hydrogels. All the solutions were prepared in DMEM, and pH was adjusted to 7.4 before mixing. The nal concentration of PAG in the gel was 6 wt%, and concentrations of hydrazide groups varied from 50 to 300 mm. All the hydrogels were immersed in DMEM (pH 7.4) at 37 C for 24 h to reach complete hydration before testing. 2.4. Determination of residual hydrazide groups after gel formation Trinitrobenzene sulfonic acid (TNBS) was utilized to determine the amount of residual hydrazide groups remaining after gel formation at each cross-linking condition. Briey, gels were lyophilized and exposed to a TNBS solution (5.76 mm) for 1 h. The mixture was ltered through a 0.22-mm lter and the ltrate was used for this assay [21]. The unreacted AAD or PAH in the gel reacts with TNBS and forms a soluble complex. The complex solution was diluted with 0.5 n HCl and its amount was spectrophotometrically determined at 334 nm. Dangling single-end hydrazide of AAD or PAH also form a complex with TNBS, and in this case the complex remains bound to the gel and will not be

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measured in the previous solution. Therefore, an excess amount of AAD was subsequently added to the ltrate to react with the remaining TNBS. The amount of complex bound to the gel was calculated by subtracting the amount of soluble complex from the total amount of TNBS initially added to the hydrogel. Subtracting the amount of unreacted and single-end hydrazide from the total amount of AAD or PAH used yields the amount of hydrazide that actually participated in gel formation, which can be converted to the reaction efciency. 2.5. In vitro degradation of PAG hydrogels PAG gels cross-linked with different amounts of either AAD or PAH were prepared, immersed in DMEM (pH 7.4), and incubated at 37 C under 5% CO2. Changes of weight, elastic modulus, and swelling degree of PAG gels were measured and normalized to their initial values before degradation. All the experiments were done under sterile condition to prevent bacterial and fungal contamination. 2.6. Measurements The elastic modulus in compression of PAG gels was measured by an MTS Bionix 100 mechanical tester (MTS Systems, France). The deformation rate was 0.5 mm/min, and the diameter of the indentor was 3.15 mm. The hydrogels were swollen in DMEM (pH 7.4) for 24 h at 37 C, excess water on the hydrogel was removed, and the gels were weighed to obtain the swelling degrees. FT-IR spectra were recorded by an Avatar 360 spectrophotometer (Nicolet, WI) using a KBr pellet method. The resolution was 2 cm1 with a repetition of 32 scans. Spectrophotometric measurements were performed using a Perkin-Elmer Lambda 12 UV/Vis spectrophotometer.

Fig. 1. (a) Chemical structure of PAG gels covalently cross-linked with AAD. (b) Synthetic scheme for creating multifunctional PAH from polyacrylamide.

Table 1 Characteristics of PAG hydrogels covalently cross-linked with different types of cross-linking molecules Hydrazide (mm) AADa E (kPa) 50 100 200 300
a b

PAHb Q n.a.c 16.370.4d 12.670.2 12.270.3 E (kPa) 5.572.9d 28.273.9 191.4717.2 236.8726.8 Q 17.771.6d 14.970.2 10.170.3 6.870.6

n.a.c 2.070.7d 18.371.5 22.570.4

Adipic acid dihydrazide. poly(acrylamide-co-hydrazide). c Not applicable. d Measured after being swollen in DMEM for 6 h, as gels were completely disintegrated in 12 h.

3. Results and discussion PAH, a multi-functional cross-linking molecule, was successfully synthesized using polyacrylamide and hydrazine (Fig. 1). The degree of substitution of hydrazide groups to acrylamide groups was 52%, as determined by measuring the complexation behavior of TNBS with hydrazide spectrophotometrically at 334 nm. PG was isolated from commercially available alginate by acid hydrolysis, and then oxidized with sodium periodate to prepare PAG. The weight-average molecular weight of PAG was 6000, and its degree of oxidation was determined to be 67% [21]. PAG was subsequently cross-linked with either a bi-functional cross-linker (AAD) or a multi-functional cross-linker (PAH). The coupling reaction was conrmed by appearance of a

carbonyl band of the hydrazide group at 1658 cm1 from the FT-IR spectra. We rst prepared PAG gels at various concentrations of either AAD or PAH, and investigated their physical properties including mechanical stiffness and swelling degree (Table 1). PAG gels were not formed with inclusion of 50 mm hydrazide from AAD, likely due to the lack of effective cross-links. PAG did, however, form gels with PAH at the same concentration (50 mm hydrazide). However, these gels disintegrated before they reached complete hydration. As the hydrazide concentration was raised, the number of effective crosslinks increased in both gels cross-linked with AAD and PAH. This resulted in the formation of stable hydrogels that exhibited an increase of the elastic modulus and a decrease of the degree of swelling in this range of hydrazide concentrations as expected. The theoretical equimolar ratio of aldehyde to hydrazide groups in the

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gel was determined to be at 200 mm hydrazide. A dramatic change in the mechanical stiffness of PAG gels cross-linked with AAD or PAH between 100 and 200 mm hydrazide can be attributed to this proximity to an equimolar ratio of both groups in the system. The gels cross-linked with PAH showed higher mechanical stiffness (about one order of magnitude in elastic moduli) than AAD-cross-linked gels at the same extent of cross-linking functional groups added to the gels. This is likely attributed to the multiple attachments of PAH in the gels that enhance the effective cross-linking density, as well as a contribution of the weight fraction of PAH (e.g., 4.5% of the hydrated gel mass at 300 mm hydrazide) and the resultant mechanical properties from the polyacrylamide backbone. This result was not due to a signicant difference in the efciency of coupling of hydrazides to aldehydes with the two cross-linkers, as the reaction efciency was high and similar in both cases over the entire hydrazide concentration range (e.g., 84.072.8 vs. 86.073.0% for AAD and PAH, respectively, at 200 mm hydrazide). We next monitored mass changes of PAG gels over time. It is generally considered that hydrazone bonds formed from the reaction between aldehydes and hydrazides are labile to hydrolysis, and this suggested PAG hydrogels should be degradable in aqueous media by a bulk erosion process. AAD-cross-linked gels with 100 mm hydrazide disintegrated before they reached complete hydration. Strikingly, PAG gels cross-linked with PAH showed signicantly retarded degradation behavior as compared to gels cross-linked with AAD (Fig. 2). PAH-cross-linked gels with 300 mm hydrazide lost only 10% of their original mass after 60 days of incubation. However, gels cross-linked with the same amount of AAD completely degraded within 30 days of

incubation. Changes of elastic moduli of the gels during degradation were also monitored. PAG gels cross-linked with AAD lost their mechanical stiffness much faster than PAH-cross-linked gels, irrespective of the same hydrazide concentrations in the gels (Fig. 3). AADcross-linked PAG gels with 300 mm hydrazide completely lost their mechanical stiffness within 20 days of incubation. However, PAH-cross-linked gels gradually lost their mechanical stiffness, and maintained almost 50% of their initial elastic moduli even after 60 days of incubation. This nding is consistent with results of mass loss. The swelling degree is strongly related to the mechanical properties of gels and reects the crosslinking density of the gels. The cross-linking density was calculated based on the theory of rubber elasticity [23], as network chains in all the gels used in this experiment follow Gaussian statistics model (Fig. 4): G cRT=Mc 1 2Mc =Mn Q1=3 ; ve r=Mc ; 1 2

where G is the shear modulus, c is the concentration of the polymer in a solution, Mc is the molecular weight between cross-links, Mn is the number-average molecular weight, Q is the degree of swelling, ne is the crosslinking density, and r is the polymer density (=0.8755 g/ cm3) [21]. Under the assumption of afne network model, the shear modulus (G) can be obtained from a slope of s vs. (l l2 ) plot, where s is the stress and l is the ratio of the deformed length to the undeformed length of the hydrogel [24]. The cross-linking density of the covalently cross-linked gels was close to that of typical calcium cross-linked alginate gels (B104 mol/ cm3). The cross-linking density of AAD-cross-linked

Fig. 2. Weight loss of PAG hydrogels during degradation. The gels were cross-linked with either AAD ((J) 200 mm hydrazide; and (&) 300 mm hydrazide) or PAH ((m) 100 mm hydrazide; (K) 200 mm hydrazide; and () 300 mm hydrazide).

Fig. 3. Change of mechanical stiffness of PAG hydrogels during degradation. The gels were cross-linked with either AAD ((J) 200 mm hydrazide; and (&) 300 mm hydrazide) or PAH ((m) 100 mm hydrazide; (K) 200 mm hydrazide; and () 300 mm hydrazide).

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Fig. 4. Log G vs. log Q plot of all hydrogels used in this experiment.

Fig. 5. Change of the cross-linking density of PAG hydrogels during degradation. The gels were cross-linked with either AAD ((J) 200 mm hydrazide; and (&) 300 mm hydrazide) or PAH ((m) 100 mm hydrazide; (K) 200 mm hydrazide; and () 300 mm hydrazide).

gels rapidly decreased during degradation of the gels. However, gels cross-linked with PAH (i.e., 200 and 300 mm hydrazide) experienced a continual decrease in the net cross-linking density during degradation (Fig. 5). The slow and continual degradation behavior of gels cross-linked with multi-functional cross-linking molecules (e.g., PAH) might be attributed to the multiple attachment points that only allow complete disintegration of the cross-linking molecules once all attachment points are lost. This is in contrast to bi-functional crosslinking molecules, which can be released following the loss of only two attachment points. Individual AAD molecules may be more likely to form intramolecular cross-links than the larger, multifunctional PAH, and this could also contribute to the more rapid degradation of AAD-cross-linked gels. Previous studies, however, indicate that the degradation of the AAD-cross-linked PAG gels is controlled by the hydrolysis of the hydrazone cross-links [21], suggesting efcient intermolecular cross-linking even with this molecule. The multifunctional cross-linking molecules may also efciently re-cross-link during the degradation, as we have previously demonstrated that gels formed with this chemistry can reversibly cross-link [21]. The continued proximity of PAH to the PAG during degradation, due to the multiple attachment points, would presumably enhance this process. This general approach to control the mechanical properties and degradation behavior of hydrogels may nd wide utility in other systems. Hydrogels from natural polymers are generally short of physical strength, and chemical cross-linking methods have been introduced to improve the mechanical properties of the gels. Small molecules with bi-, tri-, and tetra-functional groups have been often utilized to cross-link the

polymers [25,26], and the resultant gels often still exhibit a limited range of mechanical strength. Poly(ethylene glycol) is one of the most frequently used synthetic polymers in biomedical applications, and several approaches have been reported to form hydrogels. Poly(ethylene glycol)-based degradable hydrogels can be synthesized and modied with peptides inferring specic integrin-binding capability [27]. However, these gels typically exhibit low mechanical strength (Youngs modulus o3.5 kPa). Photoinitiated polymerization of poly(ethylene glycol)-derivatives may readily form gels in situ, and has been utilized to form degradable hydrogels for tissue regeneration [28]. However, some of these gels degrade very quickly, likely due to the lack of efcient cross-links. In all of these systems multifunctional cross-linkers may be useful to control the ranges of the mechanical properties and degradation rates of the gels. In summary, we have found that PAG hydrogels cross-linked with a multi-functional cross-linking molecule exhibited high mechanical stiffness, likely due to the increase of effective cross-links. This approach, to control degradation behavior and mechanical strength of hydrogels using multi-functional cross-linking molecules instead of bi-functional molecules, may be useful to develop hydrogels from natural and synthetic polymers for various biomedical applications, including delivery of drugs or cells.

Acknowledgements The authors thank the National Institutes of Health (R01 DE13033) and Curis for their nancial support.

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