Sustained Release of Human Growth Hormone from Heparin-Based

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Notes Sustained Release of Human Growth Hormone from Heparin-Based Hydrogel Won Il Choi, Mihye Kim, Giyoong Tae,* and Young Ha Kim Research Center for Biomolecular Nanotechnology and Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju, Korea, 500-712 Received December 14, 2007 Revised Manuscript Received March 23, 2008

Introduction Human growth hormone (hGH) is a protein of 191 amino acids with a molecular weight of approximately 22 kDa, it has a unique role in promoting longitudinal bone growth, and an important function in the regulation of protein, lipid, and carbohydrate metabolism.1,2 Therefore, hGH is used to treat children with short stature caused by growth hormone deficiency, Turner’s syndrome, or chronic renal failure. Also, hGH is applied in growth hormone replacement therapy for adults.3,4 The current hGH administration is by injecting subcutaneously daily or three times a week for a period of several years, thus having problems associated with short half-life, renal toxicity, and the requirement of frequent injections. So, hGH has been one of the main targets for the development of sustained release formulation.5 Previous approaches for the sustained release formulations of hGH are either by directly modifying hGH by PEGylation to decrease the rate of clearance, or by developing new encapsulation formulation to obtain the sustained release of hGH. In the latter case, research has been focused on encapsulation of hGH into physically cross-linked formulation either solid spheres like PLGA or hydrogels.3,6,7 In either cases, the sustained release of hGH is achieved by the diffusion/degradation from the matrix, not by any interaction with the matrix. Therefore, these systems still have problems such as a high initial burst or denaturation/aggregation of the loaded protein.5,8–10 The objective of this study is to demonstrate that a heparinbased hydrogel is applicable to a new hydrogel formulation for the sustained release of hGH based on the interaction between heparin and hGH. Heparin is a natural, highly sulfated, and anionic polysaccharide composed of repeating disaccharides of 1f4-linked glucosamine and uronic-acid residues. Heparin is best known for its anticoagulant properties, and also interacts with a variety of proteins that have heparin-binding domains including most of growth factors.11,12 Based on the high specific binding of heparin for various growth factors, several heparincontaining systems have been developed for the sustained release of growth factors.11,13–16 In addition, it was also reported that heparin can make complexation and precipitation with hGH.2 Therefore, it was hypothesized that a heparin-based hydrogel can be applied as a sustained release system of hGH, having binding affinity between target molecule and the matrix, in * To whom correspondence should be addressed. Telephone: 82-62-9702305. Fax: 82-62-970-2304. E-mail: [email protected].

contrast to the diffusion/degradation based-sustained release systems developed so far.3,17,18 Previously, we reported the development of a heparin-based hydrogel system with the potential to be gelled in the presence of cells or proteins by reacting thiol-functionalized heparin (Hep-SH) and diacrylated poly(ethylene glycol) (PEG-DA).19 Here, we characterized the release of hGH from this heparin-based hydrogel as a sustained release system of hGH with binding affinity. For comparison, we also characterized the release profile of hGH from the PEGbased hydrogel with similar mesh size.

Experimental Section Materials. Heparin (sodium salt, from porcine intestinal mucosa, Mw 12 kDa) was purchased from Cellsus Inc. (Cincinnati, Ohio). Poly(ethylene glycol) diacrylate (PEG-DA, Mw 3.4 and 6 kDa, degree of substitution 98%) and tetra-functional poly(ethylene glycol) sulfhydryl (PEG-SH4, Mw 10 kDa) were purchased from SunBio Inc. (Anyang City, Korea). 1-Ethyl-3-[3-dimethylamino] propyl]carbodiimide (EDC), 1-hydroxy-benzotriazole hydrate (HOBT), cysteamine, dithiotreitol (DTT), potassium phosphate monobasic, sodium phosphate dibasic, sodium azide, and tris-(hydroxymethyl) aminomethane were purchased from Sigma (St. Louis, MO). Sodium chloride and potassium chloride were obtained from Merk (Darmstadt, Germany). Phosphate-buffered saline (PBS, 0.01 mol/L PBS solution with 0.138 mol/L NaCl and 0.0027 mol/L KCl, pH 7.4) was prepared with potassium phosphate monobasic and sodium phosphate dibasic. Lysozyme was also obtained from Sigma, and bovine serum albumin was purchased from AMRESCO (Solon, Ohio). Human growth hormone (hGH) was a kind donation from LG Biotech (Seoul, Korea). Ellman’s reagent was obtained from Pierce (Rockford, IL). All chemicals were used without further purification. Sterilization syringe filters (0.2 µm) were obtained from Whatman (Florham Park, New Jersey, USA). Hydrogel Preparation via Michael-Type Addition. The heparinbased hydrogel was prepared by reacting Hep-SH and PEG-DA, as previously reported by us.17 Thiol-functionalized heparins (Hep-SH) with from 30 to 50% conversion of COOH group to thiol group were obtained. Briefly, heparin was dissolved in deionized water, and EDC, HOBt, and an excess amount of cysteamine were added. The molar ratios among reactants were varied to control the modification of carboxylic groups. After the reaction, an excess of DTT was used to reduce the oxidized disulfide groups in order to get free thiol groups. After attachment of cysteamine, H in two methylene units in cysteamine were clearly observed in 1H NMR spectra, and the intensities increase in proportion by increasing the degree of thiolation (see the Supporting Information). Due to the broad distribution of heparin spectrum in NMR, the amount of thiol groups attached to heparin was measured using the molar absorptivity of Ellman’s reagent at 412 nm. Then, Hep-SH and 6 kDa PEG-DA (1:1 molar ratio of thiol group and acrylate group) were dissolved in degassed PBS at 10.0 or 15.0% (w/v). In comparison, the PEG-based hydrogels were prepared by reacting tetra-functional poly(ethylene glycol) sulfhydryl (PEG-SH4, 10 kDa) and 3.4 kDa PEGDA (1:1 molar ratio of thiol group and acrylate group) at the same concentrations to make hydrogels with similar mesh size and mechanical properties of the heparin-based hydrogel. After 0.2 µm sterile filtration, precursor solutions were incubated at 37 °C for 1 h to induce hydrogel states. Rheological Measurements. The gelation process was monitored using a rheometer (Gemini, Malvern Instrument, England) equipped

10.1021/bm701391b CCC: $40.75  2008 American Chemical Society Published on Web 05/06/2008

Notes with a temperature controller at 37 °C and a solvent trap to suppress the drying of the polymer solution during gelation. Samples were analyzed with sandblast parallel plate geometry and a 500 µm gap thickness (the volume of sample: 0.1 mL). An angular frequency of ω ) 1 rad/s and strain of γ ) 0.1% were selected to ensure a linear regime of oscillatory deformation. Because shear rate can influence the cross-linking process,20 constant ω and γ values were used for all experiments. Swelling Studies. The swelling ratios of the heparin-based hydrogels and the PEG-based hydrogels with the same initial total concentration (10.0 or 15.0% (w/v)) were measured in PBS. Prepared hydrogels were immersed in 50 mL tube containing 20 mL PBS (pH 7.4) and then placed in an incubator at 37 °C for 2 days. Fully swollen hydrogels were weighed (Ws) immediately after the removal of excess water by rolling them in wet tissue papers. Then, the hydrogels were lyophilized and weighed (Wd). The swelling ratio was calculated by Ws/Wd (n ) 4). Protein Structure Modeling. To characterize the amino acids of hGH on its surface and electrostatic potential of hGH, hGH was visualized using RasMol software (http://www.umass.edu/microbio/ rasmol/getras.htm#raswin). Heparin Affinity to Proteins. Heparin affinity chromatography (AFpak AHR-894 affinity column (8.0 mm ID × 50 mm L, Shoko, Co., Tokyo, Japan)) was used to characterize the interaction between heparin and hGH. Lysozyme and bovine serum albumin (BSA) were used as a positive and a negative control, respectively. Two kinds of eluting buffer systems were used. First, 10 mM Tris-HCl buffer (pH 7.4) with 10 mM NaCl (buffer A) and 10 mM Tris-HCl buffer (pH 7.4) with 300 mM NaCl (buffer B) were applied by a gradient from 0 to 100% of buffer B (from 0 to 30% B for the first 20 min and from 30 to 100% B for the following 20 min) with 0.2 mL/min flow rate for 40 min. Second, deionized water (buffer A) and 10 mM Tris-HCl buffer (pH 7.4) with 300 mM NaCl (buffer B) were applied by a gradient from 0 to 100% of buffer B (from 0 to 20% B for the first 20 min, from 20 to 50% B for the next 10 min, and from 50 to 100% B for the following 10 min) with 0.2 mL/min flow rate for 40 min. The concentrations of injecting samples were ∼250 µg/mL with 100 µL injection volume. In Vitro hGH Release Experiments. hGH-loaded hydrogels (the heparin-based hydrogel with 40% thiolation Hep-SH and the PEGbased hydrogel) at 10.0% (w/v) polymer concentration with various loading amounts of hGH (hGH/polymer ratios of 1:10, 1:5, and 1:2.5 by dry weight, corresponding to the hGH concentration of 10 mg/mL for 1:10, 20 mg/mL for 1:5, and 40 mg/mL for 1:2.5) were prepared by adding hGH solution during gelation, and the release profiles of hGH from hydrogels were characterized. Briefly, precursor solutions (50 µL) were prepared by mixing 25 µL of hGH solution and 25 µL 20% (w/v) of gel forming components, and poured into a 15 mL centrifuge tube before gelation and then incubated at 37 °C for 2 h for gelation. A release medium, 10 mL PBS with 0.05% NaN3, was added to each tube, and the tube was put on a shaking rocker with 30 rpm at 37 °C. The whole release medium was replaced with the fresh one to maintain an infinite sink condition at each time points. Collected release mediums were immediately frozen at -80 °C until further analysis. The amounts of released hGH at each time points were analyzed with micro BCA Protein Assay Kit (Pierce, Bonn, Germany). Also, Hep-SH with various degrees of thiolation (30, 40, and 50%) were used to prepare the hGH-loaded heparin-based hydrogels with different cross-linking densities and different heparin modifications. In this experiment, 50 µL hydrogels with 10.0% (w/v) polymer concentration and 1:10 dry weight ratio of hGH/polymer were used, and the release profiles were analyzed. In addition, 15.0% (w/v) of the heparin-based hydrogel (using 40% Hep-SH) and the PEG-based hydrogel were prepared and characterized to investigate the concentration effect of the hydrogels on the hGH release profiles. All experiments were performed in quadruplicate.

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Size Exclusion Chromatography (SEC)-HPLC Analysis. SECHPLC was used to investigate the structural integrity of the released hGH from the heparin-based hydrogels and the PEG-based hydrogels. The released hGH samples from the hydrogels were analyzed using an aqueous size exclusion column (PROTEIN KW-800 series column (SEC, 8.0 mm ID × 300 mm L, Shoko, Co., Tokyo, Japan)): 1 mL/ min of 10 mM phosphate buffer with 30 mM of NaCl was used as a mobile phase, with absorption at 220 nm for eluted sample detection. Circular Dichroism (CD) Experiments. CD spectra of hGHs treated at different conditions were measured at 25 °C on a Jasco 810 spectropolarimeter (Jasco, MD) equipped with a temperature control unit. For far-UV CD spectra, samples with 5 µM were loaded into a 0.1 cm path-length quartz cell. Far-UV CD spectra were obtained by averaging five scans in the 250-200 nm wavelength range. The CD results were converted to mean residue ellipticity (MRE, degrees cm2 decimole-1) units considering a mean residue weight (MRW) of 116 and 22 kDa molecular weight of hGH.21,22 Denaturation of hGH was achieved by repeating the process of freezing/thawing, followed by vortexing for 5 min. Cell Culture Assay of hGH Bioactivity. Bioactivity of hGH released from the hydrogels was assayed using the hGH-dependent proliferation of adult human dermal fibroblast cells (HDF-A), according to the literature.23,24 Adult human dermal fibroblasts (Modern Cells and Tissue Technologies, Korea) were grown in a humidified atmosphere of 95% air and 5% CO2, at 37 °C in a DMEM/F-12 (3:1) mixture containing 10% FBS (fetal bovine serum; all from Gibco, NY). After a 1 day incubation of HDF-A cells (passage 9, 40000 cells/well in a 24-well plate), cells were washed with phosphate-buffered saline solution (PBS), and replaced by serum-free medium (SFM), followed by 2 day incubation. Then, native human growth hormone (hGH) and hGH released from both heparin-based hydrogel and PEG hydrogel at day 7 were added at two different concentration, 10 ng/mL and 100 ng/mL, and incubated for 1 day. The cells incubated in SFM were used as control. The effect of hGH to cells was assessed by measuring cell metabolic activity with cell proliferation reagent WST-1 assay (Roche Ltd., Basel, Switzerland).25 Briefly, 10% (v/v) of WST-1 reagent was added to each of the 24-well plates. After a 4 h incubation in the dark, the absorbance was measured at 450 nm, using a scanning multiwell spectrophotometer (FL600, Bio-Tek, VT).

Results and Discussion Network Structures of Hydrogels. For the heparin-based hydrogel preparation, thiol-functionalized heparins (Hep-SH) were obtained, and by varying the molar ratios among the reactants (HOBT, EDC, and cysteamine), the degrees of substitution of the heparin COOH group to the thiol group were systematically controlled from 30 to 50%, as previously reported.17 Then, hydrogels were formed via Michael-type addition reactions between multithiol groups and diacrylate linkers (Figure 1). Under the assumption of ideal network structure,16,26,27 the number-average molecular weight between cross-links, Mc, can be calculated using the following equation:19

Mc|initial,ideal ) 2Mr ) 2

(

) (

)

MWA MWB MWA MWB + )2 + fA fB 2 fB (1)

where MWA is the molecular weight of the PEG-DA and MWB is the molecular weight of the multiarmed thiol precursor, fA and fB are the number of reactive functionalities per acrylate and thiol-bearing species, respectively, and Mr is a parameter that represents the smallest fundamental unit or polymer chain of which the cross-linked network is composed. The molecular weight of heparin is about 12 kDa, and the dimer unit of heparin has one carboxylic group. Thus, 40%

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Notes

Figure 1. The ideal network structures of the heparin-based hydrogel and the PEG-based hydrogel. Multiarm thiols and linear PEG DA were reacted in PBS solution at physiological conditions via Michael-type addition reaction.

kDa PEG-DA and 10 kDa PEG-SH4, fA is 2 for PEG-DA and fB is 4 for PEG-SH4. Mc values of the hydrogels, calculated by eq 1, were 8.8 kDa for the heparin-based hydrogel and 8.4 kDa for the PEG-based hydrogel. Thus, two hydrogels have relatively similar mesh sizes, and the PEG-based hydrogel has a little smaller mesh size. Therefore, for the same protein loaded in the hydrogel, similar or a little slower protein release would be expected from the PEG-based hydrogel than from the heparinbased hydrogel if diffusion through the hydrogel mesh is the main release mechanism of the loaded protein. Comparison of Mechanical Properties and Swelling Ratio between Two Hydrogel Systems. Gelation kinetics and storage modulus (elastic modulus) of two hydrogel systems were characterized using a rheometer (Figure 2). By increasing the total precursor concentration of the hydrogels from 10.0 to 15.0% (w/v), the stronger gels and the faster gelation were obtained, as expected from the fact that the higher precursor concentration increases the possibility of junction formation between thiol group and acryl group. However, except the concentration effect, it could be concluded that the mechanical characteristics of two hydrogel systems, the heparin-based hydrogel and the PEG-based hydrogel, were almost same, based on the gelation kinetics (Figure 2a) as well as the final storage modulus after gelation (Figure 2b). The swelling ratios of two hydrogels in PBS showed no noticeable difference between two systems (Table 1). A modest decrease in swelling ratio was observed by increasing total Figure 2. Gelation kinetics (a) and the final gel strengths (b) of the heparin-based hydrogel and the PEG-based hydrogel. Equimolar addition between thiol and acryl groups was used for both the heparinbased hydrogel (6 kDa PEG-DA and 40% thiolated Hep-SH) and the PEG-based hydrogel (3.4 kDa PEG-DA and 10 kDa PEG-SH4). Two different total polymer concentrations (10.0 and 15.0% (w/v)) were examined.

thiolation represents about 8.7 thiol units per heparin molecule,28 namely, fA is 2 for 6 kDa PEG-DA, and fB is 8.7 for 40% HepSH. In the case of the PEG-based hydrogels prepared by 3.4

Table 1. Swelling Ratios (Ws/Wd) of the Heparin-Based Hydrogel and the PEG-Based Hydrogel at Different Initial Concentrationsa gel type initial concentration (% (w/v))

heparin gel

PEG gel

15 10

21.0 ( 1.1 25.2 ( 1.1

23.9 ( 3.7 28.9 ( 3.0

a In PBS (pH 7.4; means ( standard deviation with n ) 4). Hydrogels were prepared by the equimolar addition of functional groups of precursors at 10.0 and 15.0% (w/v). Swelling ratio was calculated by Ws (wet weight)/ Wd (dry weight).

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Figure 3. hGH structure modeling from RasMol software. (a) Basic amino acid residues of hGH (8 lysine, 10 arginine, and 3 histidine residues) and (b) surface-exposed charged residues.

precursor concentration, regardless of hydrogel type, as expected. All hydrogels had very high water content, thereby, diffusion of loaded protein inside the hydrogels can occur without much restriction in the absence of interaction between loaded protein and hydrogels. In summary, as predicted and theoretically based on the ideal network formation, two hydrogel systems at the same concentration are supposed to have similar network structure, meaning similar effective mesh size. Characterization of Heparin Affinity to hGH. To identify interaction between hGH and heparin, molecular modeling and heparin affinity chromatography were used. In the case of molecular modeling, RasMol software was used to characterize residues on the surface from 3-D structure of hGH. Protein structure modeling shows that hGH has sufficient basic amino acid residues on the surface (8 lysine, 10 arginine, and 3 histidine residues; Figure 3a) that could potentially interact with negativelycharged heparin (Figure 3b).2 Heparin affinity chromatography was used to assess the affinity of hGH to heparin. By comparing with the positivelycharged, heparin binding protein (lysozyme, pI ) 9.3) and the negatively-charged, nonbinding protein (BSA, pI ) 5.1), the affinity of hGH was characterized. As shown in Figure 4a, positively-charged lysozyme was eluted at 17.0 min by electrostatic binding with negatively-charged heparin, whereas hGH and BSA were eluted much faster than lysozyme. However,

hGH was eluted later (at 8.4 min) than BSA (at 7.6 min). By changing the eluting buffer, the difference between BSA and hGH became clearer (Figure 4b). Thus, even though it is not very strong, it is apparent that there is an interaction between heparin and hGH in aqueous environment, as predicted from protein modeling (Figure 3) as well as the reported complexation.2 In Vitro hGH Release from Heparin-Based Hydrogel. Release of hGH from both types of hydrogels in PBS showed little initial burst. However, from the beginning, hGH release from the heparin-based hydrogel was much slower than that from the PEG-based hydrogel prepared at the same polymer concentrations (Figure 5a, 10.0% (w/v) polymer, 1:10 and 1:5 ratio of hGH/polymer); complete release in a week from the PEG-based hydrogel was observed, whereas less than 50% of loaded hGH was released in a week from the heparin-based hydrogel, and the difference between two systems was clear from day 1. As discussed in the previous section, two hydrogels are supposed to have similar mesh size, thus, similar release profiles would be expected if hGH release were dominated by diffusion only. Also, considering the net negative charge of hGH (pI ) 5.2), the faster release of hGH would be expected from the negatively-charged heparin gel than from the neutral PEGbased hydrogel. Therefore, much sustained release of hGH from the heparin-based hydrogel than from the PEG-based hydrogel at physiological buffer (pH 7.4) must result from the interaction

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Figure 4. Heparin affinity chromatography of hGH, BSA, and lysozyme. (a) Elution from 10 mM Tris-HCl buffer (pH 7.4) with 10 mM NaCl (buffer A) and 10 mM Tris-HCl buffer (pH 7.4) with 300 mM NaCl (buffer B) by a gradient from 0 to 100% of buffer B (from 0 to 30% B for the first 20 min and from 30 to 100% B for the next 20 min) with 0.2 mL/min flow rate for 40 min. (b) Elution from deionized water (buffer A) and 10 mM Tris-HCl buffer (pH 7.4) with 300 mM NaCl (buffer B) by a gradient from 0 to 100% of buffer B (from 0 to 20% B for the first 20 min, from 20 to 50% B for the next 10 min, and from 50 to 100% B for the following 10 min).

between heparin and hGH, as revealed in the previous section. Although the interaction between individual heparin molecule and hGH molecule may not be very strong, a relatively high concentration of heparin molecules in the heparin-based hydrogel seems to provide sufficient interaction of hGH in the heparin-based hydrogel to induce a sustained release of loaded hGH. To analyze the loading capacity of hGH into the heparinbased hydrogel at a fixed initial polymer concentration (10.0% (w/v)), the loading amount of hGH was varied (dry weight ratio of hGH/polymer at 1:10, 1:5, and 1:2.5). As shown in Figure 5a, although no significant differences in release rate were observed for various loading amounts of hGH, a little decrease in release rate was observed by increasing the hGH amount from 1:10 to 1:5 dry weight ratio of hGH/polymer. Considering the relative interaction intensity between hGH and heparin molecules, the opposite trend would be expected. However, with higher hGH loading amounts in the heparin-based hydrogel (dry weight ratio of 1:5 or lower), the hGH-loaded hydrogels were not visually completely clear but became somewhat opaque.

Notes

Figure 5. In vitro release profiles of hGH. (a) hGH release from two hydrogel systems prepared with 10.0% (w/v) initial polymer concentration for different hGH loading amounts (hGH/polymer ) 1:10, 1:5, and 1:2.5 for the heparin-based hydrogel, and 1:10 and 1:5 for the PEG-based hydrogel). (b) hGH release from the heparin-based hydrogels prepared from Hep-SH with various degrees of thiolation (30, 40, and 50% conversion of -COOH to -SH) with the same initial polymer concentration (10.0% (w/v)) and hGH loading amount (hGH/ polymer ) 1:10). Also, hGH release from hydrogels with 15.0% (w/ v) initial polymer concentration (40% Hep-SH was used for heparinbased hydrogel). In all cases, n ) 4.

Therefore, at higher hGH loading conditions, hGH molecules seemed to be not completely soluble inside the hydrogel, thus, the apparent existence of precipitated state of hGH molecules might result in somewhat slower release of hGH from the hydrogel by increasing hGH loading amount. It should be noted that the precipitation of hGH was not found in the case of PEGbased hydrogel even at 1:5 ratio of hGH/polymer. Thus, the interaction between hGH and heparin probably accelerated the precipitation of hGH inside the heparin-based hydrogel. To see the effect of different thiolation of heparin used for the hydrogel preparation on the hGH release, the heparin-based hydrogels were prepared with Hep-SH with various degrees of thiolation (30 to 50% of thiolation of COOH groups of heparin). To eliminate the complexity associated with precipitation, hGH loading amounts were set to 1:10 mass ratio. As shown in Figure 5b, the heparin-based hydrogels with higher degree of thiolation of Hep-SH resulted in more sustained release of hGH. Previously, we demonstrated that the degree of thiolation of HepSH used for hydrogel preparation affects little on the loading capacity of heparin-binding proteins in the hydrogel except very specific binding of Hep-SH with antithrombin III.17 Thus, it

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Figure 7. CD spectra of hGH treated at different conditions. All samples were at 5 µM. Figure 6. Size exclusion chromatograms of hGH released from the heparin-based hydrogel and the PEG-based hydrogel. A total of 1 mL/min of 10 mM phosphate buffer with 30 mM of NaCl was used as a mobile phase, and absorption at 220 nm was used to detect proteins.

can be assumed that the individual interaction of Hep-SH and hGH does not change significantly by varying degree of thiolation of Hep-SH. Instead, the use of Hep-SH with different degree of thiolation leads to a different swelling ratio of the prepared hydrogel; the higher degree of thiolation of Hep-SH makes a hydrogel with the lower swelling ratio (see Table 2b in ref 17). Therefore, the hydrogel with smaller swelling ratio prepared from Hep-SH with higher degree of thiolation would result in more sustained release of loaded hGH probably due to both effects of the increased interaction with hGH from the higher local concentration of Hep-SH and the more suppressed diffusion from the reduced mesh size of the hydrogel. Also, by increasing the initial precursor concentration of the hydrogels from 10.0 to 15.0% (w/v), the more sustained release profiles of hGH were obtained (Figure 5b). Stability of hGH Release from Hydrogels. The integrity of hGH release from hydrogels were characterized using SEC analysis. There have been many reports to show a good correlation between the monomer state of hGH and the bioactivity of hGH. Thus, the characterization of monomer state of hGH by size exclusion column has been a standard protocol for the in vitro characterization of maintenance of hGH activity.10,29–31 As shown in Figure 6, the majority of hGH (over 98%) released either from the heparin-based hydrogel or the PEG-based hydrogel maintained its monomeric form at all time points, distinguishing from the deliberately denatured hGH, which contains dimeric/aggregate form. CD spectra also support the preservation of protein integrity of hGHs released either from the heparin-based hydrogel or the PEG-based hydrogel, revealing the maintenance of their original secondary structure (R helix; Figure 7); characteristic peaks at 222 and 208 nm were clearly preserved for hGHs released from hydrogels,22 whereas by repeating denaturation process, more destructed secondary structure of hGH to random coil was observed. Bioactivity of hGH released from the hydrogels was assayed using the hGH-dependent proliferation of adult human dermal fibroblast cells (HDF-A).23 As shown in Figure 8, the added hGH stimulated the cell proliferation in a dose-dependent manner in all cases. At 10 ng/mL, all three hGHs (native one and hGH released from either the heparin-based or PEG-based

Figure 8. hGH-dependent proliferation of adult human dermal fibroblast cells compared to the control (no hGH). Three kinds of hGH (native hGH, hGH released from either heparin-based, or PEG-based hydrogel in 1 week) were compared, and a WST-1 assay was used for cell proliferation. n ) 3 with standard deviation.

hydrogel in 1 week) showed almost the same effect on the increase in cellular metabolic activity. At 100 ng/mL, native hGH produced a little better response than hGH from the hydrogel, but the potential, subtle change might be associated with the sample collection process of hGH released from hydrogels (accumulation of the released hGH for 24 h at day 7). There was no difference in the increase in cellular metabolic activity stimulated by hGH released from the heparin-based hydrogel and hGH from the PEG-based hydrogel, which is a well-known system that keeps the bioactivity of loaded proteins. Therefore, based on the results of SEC, CD, and the cell culture assay, it can be concluded that the present heparin-based hydrogel could preserve the stability of loaded hGH, thus, hGH was mainly released as a bioactive state from the hydrogel.

Conclusions Sustained release of loaded hGH over two weeks without initial burst was observed from the heparin-based hydrogels. A much slower release rate of hGH from the heparin-based hydrogel than from the PEG-based hydrogel with the similar mesh size would result from the existing interaction between heparin and hGH, confirmed by heparin-affinity chromatography. The release rate could be further controlled by the use of Hep-SH with different degree of thiolation. In vitro characterization of released hGH from the hydrogel also revealed that the majority of hGH remained stable inside the hydrogel and were released as a bioactive monomeric form with their

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biological activity. Thus, combined with the fast gelation within 10 min, the present heparin-based hydrogel is a potential injectable delivery formulation of hGH. Acknowledgment. This research was supported by the Research Center for Biomolecular Nanotechnology at GIST, and also partially by a grant (code #: R01-2007-000-20493-0) from KOSEF, Korea. Supporting Information Available. 1H NMR spectrum of Hep-SH is provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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BM701391B

Sustained Release of Human Growth Hormone from Heparin-Based

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