Signal Transduction of Hyaluronic Acid−Peptide Conjugate for Formyl

Bioconjugate Chem. 2008, 19, 2401–2408

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Signal Transduction of Hyaluronic Acid-Peptide Conjugate for Formyl Peptide Receptor Like 1 Receptor Eun Ju Oh,† Jung-Wook Kim,‡ Ji-Hyun Kong,† Sung Ho Ryu,‡ and Sei Kwang Hahn*,† Department of Materials Science and Engineering and Department of Life Science, Pohang University of Science and Technology (POSTECH), Pohang, Kyungbuk 790-784, Korea. Received June 25, 2008; Revised Manuscript Received October 13, 2008

Agonistic and antagonistic peptides for formyl peptide receptor like 1 (FPRL1) receptor have been investigated as novel drug candidates for inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis. In this work, a novel protocol for the synthesis of hyaluronic acid (HA)-peptide (CWRYMVm) conjugate for FPRL1 receptor was successfully developed for further clinical applications of peptide drugs. Aminoethyl methacrylated HA (HAAEMA) was synthesized by the coupling reaction of tetrabutyl ammonium salt of HA (HA-TBA) and AEMA using benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluorophosphate (BOP) in dimethyl sulfoxide (DMSO). Then, HA-AEMA was conjugated with CWRYMVm in water via Michael addition reaction between methacrylate group of HA-AEMA and thiol group in cysteine. The formation of HA-peptide conjugate was confirmed by 1H NMR and gel permeation chromatography (GPC). The average number of conjugated peptide molecules could be controlled from 5 to 23 per single HA chain. The HA-peptide conjugate showed serum stability longer than four days. In Vitro signal transduction activity of the HA-peptide conjugate for FPRL1 receptor was confirmed from the elevated levels of phospho-extracellular signal-regulated kinase (pERK) and calcium ion in FPRL1 overexpressing RBL-2H3 cells. The partially decreased biological activity of HA-peptide conjugates by the steric hindrance of HA was recovered after its degradation by hyaluronidase treatment.

INTRODUCTION The formyl peptide receptor like 1 (FPRL1) receptor is one of the chemoattractant receptors encompassing G proteincoupled seven transmembrane domains. It is mostly expressed in phagocytic leukocytes and stimulates innate immunity, such as chemotactic migration, pro-inflammatory cytokine secretion, and degranulation (1). When inflammation and infection occur, chemotactic factors bind to specific heterotrimeric G proteincoupled receptors (GPCRs) such as FPRL1 receptor on the leukocyte surface (2). Activation of these receptors leads to directed migration, granule mobilization, and activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (3). The reactive oxygen species generated by the oxidase are important for microbial killing and for intercellular signaling in astrocytoma cell lines, neutrophils, monocytes, T cells, and HUVECs (1, 3). Several agonistic and antagonistic peptide sequences for the FPRL1 receptor have been investigated as drug candidates for inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis (4-6). Trp-Lys-Tyr-Met-ValDMet (WKYMVm) is one of the widely studied agonist peptides that selectively binds to and activates the FPRL1 receptor in FPRL1 overexpressing RBL-2H3 cells (4, 5). Trp-Arg-Trp-TrpTrp-Trp (WRWWWW) is an FPRL1 antagonist peptide that down-regulates the activation of FPRL1 by agonistic peptides, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, superoxide generation, and chemotactic migration of cells toward agonistic peptides (6). Recently, the bioconjugation technology using synthetic and natural polymers like poly(ethylene glycol) [PEG] and hyaluronic acid (HA) has been widely used for the development of * Corresponding author. S. K. Hahn, Ph.D. Tel.: +82-54-279-2159; Fax: +82-54-279-2399; E-mail address: [email protected]. † Department of Materials Science and Engineering. ‡ Department of Life Science.

various biopharmaceuticals with feasible pharmacokinetic characteristics (7). The chemical attachment of PEG to the biopharmaceuticals, such as protein and peptide drugs, has been reported to increase the drug efficacy by reducing renal clearance, decreasing the immunoresponse and alleviating the enzymatic degradation in the body. There are several commercialized PEGylation products, such as Neulasta (pegfilgrastim) by Amgen, Somavert (pegvisomant) by Pfizer, PEGASYS (peginterferon alfa-2a) by Roche, and so on. However, the negative effect of PEGylation has been also reported (8, 9). A repeated injection of PEGylated liposomes has been reported to result in their diminished long-circulating characteristics by a so-called “accelerated blood clearance” (ABC) phenomenon (8). In addition, PEGylation of glucagon like peptide - 1 (GLP-1) has been reported to bring about a significant decrease in its cAMP activity (9). The branch-type PEGylation with a molecular weight of 43 000 Da caused even greater biological activity loss (9). As an alternative to replace the roles of PEG, HA has been investigated as a novel drug carrier for various protein and peptide drugs (10-13). HA is a natural linear polysaccharide composed of alternating disaccharide units of D-glucuronic acid and N-acetyl-D-glucosamine with β(1f4) interglycosidic linkage (10). HA is the only nonsulfated glycosaminoglycan (GAG) which is abundant in synovial fluid and extracellular matrix (ECM) (10). The fact that HA molecules from different sources have the same primary structure explains the molecular basis for its natural biocompatibility (14). HA plays important roles in the regulation of cell behavior including cell migration and proliferation (15-18). CD44 (19), RHAMM (15, 20), and LYVE-1 (21) have been identified as HA receptors. Because of the various biological functions and unique physicochemical properties, HA and modified HA have been widely used for drug delivery (11, 12), arthritis treatment (22), ophthalmic surgery (23), and tissue engineering (24). Especially for drug delivery applications, HA with a high molecular weight over 2 million was used for the

10.1021/bc800255y CCC: $40.75  2008 American Chemical Society Published on Web 11/14/2008

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sustained-release formulation of human growth hormone (11), and selectively cross-linked HA hydrogels were used for the encapsulation of erythropoietin (12). HA was also used for the conjugation with active cytotoxic agents, such as paclitaxel (25) and doxorubicin (26). In this work, a novel bioconjugation protocol for FPRL1 peptide therapeutics was developed to make them suitable for in ViVo applications. Bioconjugation of the peptide drug to HA increases its half-life in circulation contributing for a high efficacy. In contrast to PEGylation, HA can be conjugated with various numbers of peptide drug molecules per single HA chain, which enables multiple action of peptide drugs. The peptide drug used in this study was one of the agonistic peptides for FPRL1 receptor with a sequence of WRYMVm. In order to introduce a thiol group to the peptide molecule, cysteine was added to the end of the peptide sequence. Aminoethyl methacrylated HA (HA-AEMA) was newly synthesized in DMSO using the tetra-n-butylammonium salt of HA (HA-TBA) and benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP), and N,N-diisopropylethylamine (DIPEA). CWRYMVm was conjugated to HA-AEMA via the Michael addition reaction between the methacryloyl group of HA-AEMA and the thiol group in cysteine. Michael addition, between electron-poor olefins and nucleophiles such as thiols, has been used for the bioconjugation of various biomolecules (27, 28). The stability of peptide in fetal bovine serum (FBS) was investigated before and after its conjugation to HA. Since the binding of agonistic peptide to FPRL1 receptor induces the elevation of phospho-extracellular signal-regulated kinase (pERK) and calcium ion levels (1), the biological activity of HA-peptide conjugates was assessed from the intracellular level of pERK and calcium ion in FPRL1 overexpressing RBL-2H3 cells. RBL2H3 cells have no FPRL1 showing similar characteristics to the other immune cells with FPRL1. Therefore, FPRL1 overexpressing RBL-2H3 (RBL-2H3/FPRL1) cells are a very good model system to check the FPRL1 downstream signaling such as pERK level.

MATERIALS AND METHODS Materials. Sodium hyaluronate, the sodium salt of hyaluronic acid (HA), with a molecular weight of 200 000 was obtained from Denkikagaku Kogyo Co. (Tokyo, Japan). Peptide with a sequence of Cys-Trp-Arg-Tyr-Met-Val-DMet (CWRYMVm) was purchased from Peptron (Daejeon, Korea). Dowex 50WX840 ion-exchange resin, benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate (BOP), 2-aminoethyl methacrylate hydrochloride (AEMA), N,N-diisopropylethylamine (DIPEA), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), trifluoroacetic acid (TFA), and hyaluronidase from Streptomyces hyalurolyticus were purchased from Sigma-Aldrich (St. Louis, MO). Tetra-n-butylammonium hydroxide (TBA-OH) was obtained from Alfa Aesar (Ward Hill, MA). Dimethyl sulfoxide (DMSO) was obtained from Junsei Chemical Co. (Tokyo, Japan) and acetonitrile from J. T. Baker (Phillipsburg, NJ). Anti-rabbit polyclonal antibody to phospho-ERK was purchased from Cell Signaling Technology (Danvers, MA) and anti-mouse monoclonal antibody to GAPDH from Biogenesis (Poole, UK). Goat anti-rabbit IgG or goat anti-mouse IgG antibody conjugated to horseradish peroxidase was obtained from KPL (Gaithersburg, MD). Double-distilled water was used for the following experiments. All chemicals were used without further purification. Synthesis of HA-AEMA. Ion exchange resin of Dowex 50WX-8-400 (25 g) was washed with 500 mL of water and filtered to remove the supernatant three times. Then, 1.5 molar excess of TBA-OH (48.9 mL) was added to the Dowex resin and mixed for 30 min. The filtered Dowex-TBA resin was

Oh et al.

washed with 500 mL of water and filtered again three times. Sodium salt of HA (MW ) 200 K, 5 mmol) was dissolved in 200 mL of water, which was poured into the prepared DowexTBA (25 mmol) resin. After mixing for 3 h, the supernatant was filtered through 0.45 µm filter and lyophilized for 3 days. The resulting HA-TBA was dissolved in DMSO. Then, BOP, 2-AEMA and N,N-DIPEA were added to the solution and mixed for a day. Finally, the reaction product was dialyzed against a large excess amount of water and lyophilized for three days. The obtained HA-AEMA was characterized with 1H NMR (NMR, DPX300, Bruker, Germany). Synthesis of HA-Peptide Conjugates. Agonistic peptide for FPRL1 receptor (CWRYMVm) was dissolved in water. For the reduction of the disulfide bond between peptide molecules, 10-fold molar excess of TCEP as a reducing reagent was added to the peptide solution and mixed for 10 min. HA-AEMA was also dissolved in water. After complete dissolution, the HAAEMA solution was mixed with the peptide solution. The number of peptide molecules per single HA chain in the feed was 5, 9, 19, 28, and 56, respectively. After the pH of the reaction mixture was adjusted to 8.74 by the addition of 1 N NaOH, the mixed solution was incubated at 37 °C for 16 h. Then, the reaction was stopped by dropping the pH to 7.0 with 1 N HCl. The solution was finally lyophilized for three days. HA-peptide conjugate was purified by fractionation using gel permeation chromatography (GPC). GPC analysis was performed using the following systems: Waters 1525 binary HPLC pump, Waters 2487 dual λ absorbance detector, Waters 717 plus autosampler, Superdex Peptide 10/300 GL column. The eluent was 30 vol % acetonitrile (ACN)/0.1 vol % trifluoroacetic acid (TFA), and the flow rate was 1 mL/min. The detection wavelengths were 210 nm for HA and 280 nm for the peptide, respectively. Three replicates were carried out to assess the average peptide content in HA-peptide conjugate and the bioconjugation efficiency (%). Quantification of Peptide Content in HA-Peptide Conjugates. A peptide stock solution at a concentration of 1 mg/ mL was used to prepare peptide standard solutions with concentrations of 10, 20, 40, 80, 160, and 320 µg/mL, respectively. HA-peptide conjugate solutions were also prepared by dissolving 1 mg of each HA-peptide conjugate sample in 1 mL of water. GPC analysis was carried out as described above. From the peak areas detected at 280 nm, a linear standard curve for peptide was obtained and used for the determination of the amount of peptide in HA-peptide conjugates. In Vitro Serum Stability Test of HA-Peptide Conjugates. In order to investigate the effect of HA conjugation on the serum stability of peptide, raw peptide and three HA-peptide conjugates were dissolved in 0.5 mL of water and mixed with 0.5 mL of fetal bovine serum (FBS), respectively. In HA-peptide conjugates, the number of peptide molecules per single HA chain was 5, 19, and 33, respectively. Because the peptide was not dissolved in serum completely, 50 vol % serum solution was used for the serum stability test. Then, the solutions were incubated at 37 °C for 96 h. The remaining amount of peptide was measured by GPC analysis after incubation for 12, 24, 48, 72, and 96 h. Three replicates were carried out. Hyaluronidase Treatment of HA-Peptide Conjugates. In order to investigate the effect of HA conjugation on the signal transduction activity of peptide, three kinds of peptide samples were prepared, the raw peptide, HA-peptide conjugates, and HA-peptide conjugates after hyaluronidase treatment. In the HA-peptide conjugates, the number of peptide molecules per single HA chain was 5, 8, and 23, respectively. Each HA-peptide conjugate sample (0.4 mL, 1 mg/mL) was divided into two aliquots. One aliquot was mixed with 0.2 mL of hyaluronidase solution (400 units/mL), and the other was mixed with 0.2 mL

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Figure 1. Schematic representations (a) for the synthesis of aminoethyl methacrylated hyaluronic acid (HA-AEMA) using N,N-diisopropylethylamine (DIPEA) and benzotriazol-1-yloxy-tris(dimethyl-amino)phosphonium hexafluorophosphate (BOP) and (b) for the conjugation of HA-AEMA with the peptide (CWRYMVm) for formyl peptide receptor like 1 (FPRL1) receptor.

of water. Then, the solutions were incubated at 37 °C overnight for the complete enzymatic degradation of HA. Three replicates were carried out for the following bioactivity tests measuring the intracellular level of pERK and calcium ion. RBL-2H3 and RBL-2H3/FPRL1 Cell Cultures for Western Blot. RBL-2H3 cells and FPRL1 overexpressing RBL-2H3 (RBL-2H3/FPRL1) cells were cultured at 37 °C in a humidified incubator containing 5% CO2, as described elsewhere (29). Dulbecco’s modified Eagle’s medium (DMEM) was supplemented with 20 vol % heat-inactivated FBS and 200 µg/mL of G418. The cells were subcultured every 3 days. Stimulation of RBL-2H3/FPRL1 Cells with Peptide Samples. The prepared cells were aliquoted into 1 × 106 cells and stimulated with a control (no treatment), peptide (CWRYMVm), HA-AEMA, the mixture of peptide and HA-AEMA, and three HA-peptide conjugate samples with and without hyaluronidase treatment. For comparison, the peptide content in HA-peptide conjugate samples was adjusted to have the same amount as the peptide sample. The stimulation time varied from 0 to 30 min. After stimulation, the cells were washed with serum-free DMEM and lysed in lysis buffer at pH ) 7.4 containing 0.5 M NaCl, 20 mM Tris-Cl, 0.25% Triton X-100, 1 mM EDTA, 1 mM EGTA, 10 mM β-glycerphosphate, 10 mM NaF, 1 mM benzamidine, 10 µg/mL aprotinin, 10 µg/mL

leupeptin, 10 µg/mL pepatatin, 1.5 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM DTT. The detergent-insoluble material was pelleted by centrifugation at 12 000 g and 4 °C for 15 min, and the soluble supernatant fraction was used immediately or stored at -80 °C before use. Protein concentrations in the lysates were determined by Bradford protein assay. Three replicates were carried out. Electrophoresis and Immunoblot Analysis for Western Blot. Protein samples were subjected to electrophoresis using 12 wt % SDS-polyacrylamide gel and the buffer system described by King and Laemmli (30). After the electrophoresis, the proteins were blotted onto nitrocellulose membrane and blocked by incubating with Tris-buffered saline, 0.05% Tween 20 containing 5% nonfat dried milk. Then, the membranes were incubated for 12 h with anti-rabbit polyclonal antibody to pERK (1/1000 dilution) or anti-mouse monoclonal antibody to GAPDH (1/2000 dilution) and washed with Tris-buffered saline. After incubating the membrane with a 1/5000 diluted goat anti-rabbit IgG or goat anti-mouse IgG antibody conjugated to horseradish peroxidase for 1 h, the antigen-antibody complexes were visualized by using the enhanced chemiluminescence (ECL) detection system. The phosphorylation of pERK was analyzed using the Image J 1.38 X program.

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Figure 2. 1H NMR spectra of (a) aminoethyl methacrylated hyaluronic acid (HA-AEMA) and (b) HA-peptide conjugate of HA-(CWRYMVm)23 for formyl peptide receptor like 1 (FPRL1) receptor.

Calcium Imaging of the Cells. The intracellular calcium level was determined using fura-2-acetoxymethyl ester (Fura2/AM) as described by Bae et al. (31). Briefly, the prepared cells were incubated in serum-free RPMI 1640 medium with 3 µM of Fura-2/AM at 37 °C for 50 min under continuous stirring. For each measurement, 2 × 106 cells were aliquoted in Ca2+free Locke’s solution (154 mM NaCl, 5.6 mM KCl, 1.2 mM MgCl2, 5 mM HEPES, pH 7.3, 10 mM glucose, and 0.2 mM EGTA). Changes in the fluorescence ratio were measured at the dual excitation wavelengths of 340 and 380 nm and emission wavelength of 500 nm, and the fluorescence images were also obtained. Statistical Analysis. The data are expressed as means ( SD from several separate experiments. Statistical analyses and comparisons were carried out via t-test, and a value for p < 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Synthesis of HA-Peptide Conjugates for FPRL1 Receptor. The chemical modification of HA with AEMA was successfully carried out using the novel protocol as schematically

shown in Figure 1a. HA-AEMA was synthesized by the coupling reaction of TBA salt of HA (HA-TBA) with AEMA using BOP in DMSO. DIPEA was used to release the free primary amine of AEMA. The chemical structure of HA-AEMA was analyzed by 1H NMR. The methyl resonance of acetamido moiety of HA at δ ) 1.85-1.95 ppm was used as an internal standard (γ in Figure 2). The degree of AEMA modification was ca. 57 mol %, which was determined from the peak areas of methacrylate unit of AEMA at δ ) 6.1 and 5.6 ppm (R1 and R2 in Figure 2a). By changing the amount of AEMA added for the reaction, we could control the degree of AEMA modification in HA-AEMA up to 85 mol %. The peptide for FPRL1 receptor having a sequence of WRYMVm was modified with cysteine to prepare CWRYMVm with thiol groups. HA-AEMA was conjugated with CWRYMVm via the Michael addition reaction between methacryloyl groups in HA-AEMA and thiol groups in CWRYMVm (Figure 1b). 1H NMR analysis of HA-peptide conjugate, HA-(CWRYMVm)23, confirmed the formation of HA-peptide conjugate (Figure 2b). Before conjugation, there were double peaks at δ ) 6.1 and 5.6 ppm corresponding to two hydrogens on methacryloyl groups of HA-AEMA (R1 and

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Figure 3. Gel permeation chromatograms (GPC) of the peptide (CWRYMVm) for formyl peptide receptor like 1 (FPRL1) receptor at a right peak and hyaluronic acid (HA)-peptide conjugate at a left peak detected at 280 nm.

Figure 4. The content of peptide (CWRYMVm) for formyl peptide receptor like 1 (FPRL1) receptor in hyaluronic acid (HA)-peptide conjugates (the number of peptide molecules per single HA chain) (•, W) and the resulting bioconjugation efficiency (%) (O, w). The results are presented as means ( SD of three independent experiments.

R2). After Michael addition reaction, the peaks at δ ) 6.1 and 5.6 ppm disappeared and the peak corresponding to -CH3 of the methacryloyl group at δ ) 1.8 ppm (β) was shifted to the peak at δ ) 1.3 ppm (β′). The results from 1H NMR analysis corroborated the successful formation of HA-AEMA and the subsequent HA-peptide conjugate. Characterization of HA-Peptide Conjugates for FPRL1 Receptor. The formation of HA-peptide conjugate for FPRL1 receptor was also confirmed by GPC analysis as shown in Figure 3. The peak of HA-peptide conjugate appeared at a retention time of 8 min, while that of intact peptide appeared at a retention time of 13 min. The peak shift to the early retention time revealed that the peptide was conjugated to HA with a high molecular weight of 200 000 Da. The number of peptide molecules added per single HA chain in the reaction solution for the synthesis of HA-peptide conjugate was 5, 9, 19, 28, and 56, respectively. The resulting peptide contents in HA-peptide conjugate were quantified by measuring the peak area on GPC detected at 280 nm. Because HA is not detected at a wavelength of 280 nm, the peaks of HA-peptide conjugates at 280 nm resulted solely from the peptide molecules. The peptide content in HA-peptide conjugates at a retention time of 8 min increased with the feeding ratio of peptide molecules

Figure 5. In Vitro serum stability of peptide (CWRYMVm, b) and hyaluronic acid (HA)-peptide conjugates in fetal bovine serum (FBS, 50 vol %) solution. The number of peptide molecules per single HA chain was 5 (O), 19 (1), and 33 (0), respectively. The results are presented as means ( SD of three independent experiments.

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Figure 6. (a) Western blots for phospho-extracellular signal-regulated kinase (pERK) levels in the RBL-2H3 cells and formyl peptide receptor like 1 (FPRL1) overexpressing RBL-2H3 cells after stimulation for 5 min with a control (no treatment), peptide (CWRYMVm), aminoethyl methacrylated hyaluronic acid (HA-AEMA), and HA-peptide conjugate of HA-(CWRYMVm)23 having 23 peptide molecules per single HA chain. (b) Western blots for pERK levels in the FPRL1 overexpressing RBL-2H3 cells after stimulation for 5 min with a control (no treatment), HA-AEMA, peptide (CWRYMVm), a mixture of HA-AEMA and peptide (HA + CWRYMVm), and three HA-peptide conjugate samples (HA-CWRYMVm) with and without hyaluronidase (HAse) treatment. The numbers 5, 8, and 23 represent the number of peptide molecules per single HA chain in HA-peptide conjugates. (c) Densitometry of Western blot bands for the samples in (b). The results were presented as means ( SE of three independent experiments.

to single HA chain (Figure 4). However, the degree of bioconjugation (%) decreased with increasing peptide content in the reactants. The bioconjugation (%) represents the molar ratio of peptide molecules in the HA-peptide conjugate to the total peptide molecules added initially for the conjugation reaction. When the number of peptide molecules per single HA chain in the feed was less than 19, the bioconjugation efficiency (%) was higher than 90%. The average number of conjugated peptide molecules could be controlled from 5 to 23 per single HA chain, as schematically shown in Figure 1b. In Vitro Serum Stability of HA-Peptide Conjugates for FPRL1 Receptor. Although several agonistic and antagonistic peptides for FPRL1 receptor have been identified in our group as

promising new drug candidates for the inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis (4-6), their short halflives in the body should be elongated for further clinical applications. As expected, the peptide with a sequence of CWRYMVm was quite unstable in serum. After incubation in 50 vol % FBS solution for 3 days, only less than 30 wt % of the peptide remained with a big initial degradation of over 50 wt % in a day (Figure 5). However, HA-peptide conjugate showed notably increased serum stability in 50 vol % FBS solution. The peptide conjugated to HA did not degrade at all even after incubation for 4 days. The increased stability of peptide in serum by HA conjugation was the same for all three HA-peptide conjugates with different peptide contents. As the serum stability of the peptide drug is essential for the

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Figure 7. Calcium fluorescence imaging of the formyl peptide receptor like 1 (FPRL1) overexpressing RBL-2H3 cells after stimulation with (a) a control (no treatment) and (b) HA-peptide conjugate sample of HA-(CWRYMVm)23 for FPRL1 receptor. The number of peptide molecules per single HA chain was 23.

application in ViVo, the protocol for the conjugation of peptide to HA via Michael addition would be usefully exploited for the development of peptide therapeutics with good solubility and a feasible long half-life. In Vitro Signal Transduction of HA-Peptide Conjugates for FPRL1 Receptor. The signal transduction activity of HA-peptide conjugate for the FPRL1 receptor was assessed by measuring the elevation level of phospho-extracellular signalregulated kinase (pERK) (Figure 6) and calcium ion (Figure 7) in RBL-2H3/FPRL1 cells. The band intensity was normalized to GAPDH to get the pERK level. When the cells were stimulated with the peptide or HA-peptide conjugate for FPRL1 receptor up to 30 min, the elevation of intracellular pERK level on Western blot was the highest at a stimulation time of 5 min in both cases. Therefore, the stimulation time was fixed at 5 min for the following cell activity tests. Figure 6a compares the pERK levels on Western blots in RBL-2H3/FPRL1 cells with those in RBL-2H3 cells after stimulation with a control (no treatment), peptide (CWRYMVm), HA-AEMA (HA), and HA-peptide conjugate having 23 peptide molecules per single HA chain. As there is no receptor for CWRYMVm in RBL-2H3 cells, the pERK levels were not elevated at all even after stimulation with peptide alone. However, in RBL2H3/FPRL1 cells, the pERK levels elevated significantly after stimulation with both peptide alone and HA-(CWRYMVm)23 samples. Figure 6b shows the pERK levels on Western blots after stimulation with a control (no treatment), HA-AEMA (HA), peptide (CWRYMVm), a mixture of HA-AEMA and peptide (HA + CWRYMVm), HA-peptide conjugates (HA-CWRYMVm), and hyaluronidase-treated HA-peptide conjugates (+HAse). The numbers 5, 8, and 23 represent the number of peptide molecules per single HA chain in HA-peptide conjugates. As a control, the pERK level by peptide alone was set to be 100% and the other data were normalized for comparison (Figure 6c). In the mixture of HA-AEMA and peptide, the pERK level decreased to ca. 63% compared with that of peptide alone. Moreover, the bioactivity of HA-peptide conjugates decreased to ca. 20%, 28%, and 38% depending on the amount of peptide per single HA chain. However, the bioactivity could be recovered up to ca. 76% after degradation of HA molecules by hyaluronidase treatment (+HAse). The decrease in the signal transduction activity of peptide molecules after being conjugated to HA might have resulted from the “steric hindrance” of HA chain reducing the access of peptide molecules to FPRL1 receptor. For the same reason, HA-peptide conjugate with a low peptide content of 5 per single HA chain [HA(CWRYMVm)5] showed a lower pERK level than HA-(CWRYMVm)23, despite using the same peptide content for the Western blot analysis. After the hyaluronidase treatment (+HAse), however, the

steric hindrance was thought to be alleviated, contributing to the increase of intracellular pERK level. There was a positive correlation between the pERK level and the content of peptide in HA-peptide conjugates with and without hyaluronidase treatment. According to the t-test, the p-value between HA-AEMA (HA) and [HA-(CWRYMVm)5 + HAse] was 0.194, which means no significant elevation of pERK level after treatment with [HA(CWRYMVm)5 + HAse]. However, as the content of peptide in HA-peptide conjugates increased to 9 and 23, the p-value decreased to 0.056 and 0.004, respectively. The decreased p-value indicates that the biological activity of HA-CWRYMVm after hyaluronidase treatment significantly increased with the peptide content in HA-CWRYMVm conjugates. Bioconjugation of multiple peptides to a single HA chain might contribute to the development of long-acting formulation of peptide therapeutics. Previously, we have reported the effect of chemical modification of HA on its degradation (32, 33). The chemical modification of carboxyl groups on HA was thought to make it more stable to enzymatic degradation by hyaluronidase contributing to the elongation of its half-life. The half-life of HA-peptide conjugate will be assessed by in ViVo tests for further applications. Calcium imaging showed the same tendency as the Western blot analysis. Unlike the control, the RBL-2H3/FPRL1 cells treated by HA-peptide conjugate showed a bright fluorescence under UV light demonstrating its biological activity (Figure 7). Altogether with the stimulation test results of RBL-2H3/FPRL1 cells with agonistic peptides and HA-peptide conjugates, we could confirm the signal transduction activity of HA-peptide conjugates for the FPRL1 receptor. The novel protocol for the conjugation of HA-AEMA with the peptide of CWRYMVm would be usefully applied for various protein and peptide drugs with thiol groups. The HA-peptide conjugate for the FPRL1 receptor will be investigated further in murine models of inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis for clinical applications to immunotherapeutics.

CONCLUSIONS HA-AEMA was successfully synthesized and conjugated with CWRYMVm, one of the agonistic peptide drugs for the FPRL1 receptor, via Michael addition between the methacryloyl group of AEMA and the thiol group of cysteine. The formation of the HA-peptide conjugate was confirmed by 1H NMR and GPC. HA-peptide conjugate could be prepared to have 5-23 peptide molecules per single HA chain by changing the initial feed ratio of peptide to HA repeat unit. The conjugation of peptide to HA molecules resulted in significantly enhanced serum stability. The signal transduction activity of HA-peptide

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conjugate was confirmed by measuring the elevation level of pERK and calcium ion in FPRL1 overexpressing RBL-2H3 cells. The bioconjugation protocol using HA derivatives might be successfully applied for the development of peptide therapeutics with an elongated half-life and a feasible biological activity. In ViVo tests of HA-peptide conjugate for FPRL1 receptor will be carried out in murine models of inflammatory diseases such as sepsis, asthma, and rheumatoid arthritis for further clinical applications to immunotherapeutics.

ACKNOWLEDGMENT This study was supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080711).

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