HyaluronateâEpidermal Growth Factor Conjugate for Skin Wound

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Hyaluronate - Epidermal Growth Factor Conjugate for Skin Wound Healing and Regeneration Hyemin Kim, Won Ho Kong, Keum-Yong Seong, Dong Kyung Sung, Hyeonseon Jeong, Jin Kon Kim, Seung Yun Yang, and Sei Kwang Hahn Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01216 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 25, 2016

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Hyaluronate - Epidermal Growth Factor Conjugate for Skin Wound Healing and Regeneration

Hyemin Kima, Won Ho Konga, Keum-Yong Seongb, Dong Kyung Sungc, Hyeonseon Jeonga, Jin Kon Kimd, Seung Yun Yangb,*, Sei Kwang Hahna,*

a

Department of Materials Science and Engineering, Pohang University of Science and Technology (POSTECH),

77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk 790-784, Republic of Korea b

Department of Biomaterials Science, Life and Industry Convergence Institute, Pusan National University,

1268-50, Samnangjin-ro, Samnangjin-eup, Miryang, Gyeongnam 50463, Republic of Korea c

Department of Pediatrics, Samsung Medical Center, School of Medicine, Sungkyunkwan University, 81 Irwon-

ro, Gangnam-gu, Seoul 06351, Republic of Korea d

National Creative Research Initiative Center for Smart Block Copolymers, Department of Chemical

Engineering, POSTECH, 77 Cheongam-ro, Nam-gu, Pohang, Kyungbuk 790-784, Republic of Korea

CORRESPONDING AUTHOR FOOTNOTE *Corresponding author. Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail address: [email protected] (S. K. Hahn). Tel.: +82 55 350 5382; Fax: +82 55 350 5389; E-mail address: [email protected] (S. Y. Yang).

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ABSTRACT Epidermal growth factor (EGF) has been recognized as an excellent wound healing agent due to its therapeutic function stimulating skin cell growth, proliferation and differentiation. However, the transdermal delivery of EGF poses a significant challenge due to its short half-life and lack of efficient formulation. Here, to improve the transdermal delivery efficiency, EGF was conjugated to hyaluronate (HA), which was formulated into a patch-type film for skin wound healing. HA-EGF conjugate was synthesized by coupling reaction between aldehyde-modified HA and N-terminal amine group of EGF to minimize the loss of biological activities. The HA-EGF conjugates exhibited similar biological activities with native EGF as confirmed by ELISA and proliferation tests using murine and human fibroblasts. For the efficient topical delivery, HA-EGF conjugates were incorporated into a matrix film of high molecular weight HA. Two-photon microscopy clearly visualized more efficient transdermal delivery of HA-EGF conjugates to both normal skin and peripheral tissues around the wound area rather than that of EGF. Optical imaging and ELISA after in vivo transdermal delivery showed that the conjugation of EGF to HA retarded its degradation and extended its residence time in the wound area. Furthermore, in vivo transdermal delivery of HA-EGF conjugate in the patch-type HA film resulted in significantly improved regeneration of skin tissues even into hypodermis. [Keywords] Hyaluronate; Epidermal growth factor; Conjugate; Wound healing; Transdermal delivery


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1. INTRODUCTION Epidermal growth factor (EGF) family members, including EGF itself, heparin-binding EGF, and transforming growth factor-α, have been considered as key regulators for cell growth, proliferation, and differentiation.1,2 EGFs can bind to the extracellular domain of EGF receptor (EGFR) on the cell surface, and the concomitant intracellular signal transduction results in DNA synthesis and cell proliferation.2-4 Due to its outstanding function on the acceleration of epidermal regeneration, EGF has been widely investigated as a wound healing agent for the treatment of surgical wounds, burns and diabetic ulcers.5-7 It has been suggested that the long-term retention of EGF on wound area is favorable to form EGF-EGFR complex at the cell surface, enhancing the healing effect of EGF.8 However, topically administered EGF in the form of ointments9,10 or liquid formulations5 was difficult to place on the moisturized wound area during the healing process and the delivered EGF was easily degraded in the proteolytic environment of wounds.8,11 Accordingly, many research efforts have been devoted for developing an efficient EGF delivery system to increase bioavailability and therapeutic effect of EGF. For example, polymer patches12, hydrogels13,14, and nanofibers15 have been developed for long-term controlled delivery of EGF. These formulations were effective to modulate the release kinetics of EGF in the wound, but resulted in the limited bioavailability of EGF. Thus, there is a huge biomedical unmet need to develop EGF delivery systems with improved stability as well as long-term release for facilitated wound healing. Alternatively, polymer conjugation can be exploited to increase the stability of EGF. We have chosen a naturally-occurring linear polysaccharide of hyaluronate (HA) as a drug delivery carrier. HA has been used for a wide range of applications for drug delivery and tissue engineering.16,17 Particularly, low molecular weight (MW) HA fragments between 100 and 300 kDa promote wound repair and increase the self-defense of skin epithelium.18,19 In addition, it has been reported that majority of skin cells including fibroblasts and keratinocytes express CD44, one of HA receptors, and HA can actively participate in the regulation of skin cell proliferation by interacting with the receptor.20,21 HA also provides high permeability into skin tissues.22 HA has a hydrophobic patch domain consisting of eight CH groups, which can interact with lipid components in stratum corneum during penetration and disrupt skin barriers.22,23 Recently, we demonstrated the feasibility of HA for transdermal delivery of drugs such as protein24,25, peptide26, and nanomaterials27,28. HA conjugates showed


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significantly improved transdermal delivery of biomolecules into deep dermal tissues, and the conjugates exhibited great effect on the transdermal immunization25,26 and the treatment of skin diseases26-28 through efficient interaction with HA receptors. In this work, we have developed HA-EGF conjugates incorporated into a patch-type film of HA for efficient wound healing by facilitating its transdermal delivery into skin tissues and its facilitated interaction with skin cells. EGF was conjugated to aldehyde-modified HA (HA-ALD) at a low pH for preferred binding to N-terminal amine groups to maintain its biological activity. After characterization of the synthesized HA-EGF conjugate by gel permeation chromatography (GPC) and enzyme linked immunosorbent assay (ELISA), the biological activity before and after conjugation was assessed in murine and human fibroblasts. HA-EGF conjugates were incorporated into the patch-type film for long-term activity of EGF and convenient application to patients. Our previous transdermal delivery systems using HA were prepared in an aqueous formulation.24-28 The patch-type HA-EGF conjugates were delivered into skin tissues in a manner of controlled release and the penetration of HA-EGF conjugates was visualized by two-photon fluorescence microscopy. We assessed and discussed the long-term stability of HA-EGF conjugates against the protease-upregulated environment of wound area for up to 16 h post-treatment and the dramatic therapeutic effect of the conjugates on biopsy punch-induced wound model for further applications.


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2. EXPERIMENTAL SECTION 2.1. Materials Sodium hyaluronate (HA) with a molecular weight (MW) of 200 kDa was purchased from Lifecore Biomedical (Chaska, MN). Recombinant human EGF was obtained from Genscript (Piscataway, NJ). Sodium periodate, sodium cyanoborohydride, ethyl carbazate, tert-butyl carbazate, human serum, and fluorescein isothiocyanate isomer I (FITC) were obtained from Sigma-Aldrich (St. Louis, MO). Illustra NAPTM-5 column was purchased from GE Healthcare (Buckinghamshire, UK). Coomasie Plus (Bradford) protein assay reagent was purchased from Thermo Scientific (Rockford, IL) and human EGF ELISA kit was obtained from Peprotech (Rocky Hill, NJ). Polydimethylsiloxane (PDMS, Sylgard 184) was purchased from Hana Technology (Anyang, Korea). Balb/3T3 cells were purchased from Korean Cell Line Bank (Seoul, Korea). Primary human dermal fibroblasts were purchased from the American Type Culture Collection (ATCC, Manassas, VA). Dulbecco’s modified eagle’s medium (DMEM) was purchased from Mediatech (Manassas, VA). Rat tumor necrosis factor-α (TNF-α), and interleukin-1 (IL-1) ELISA kits were purchased form eBioscience (Vienna, Austria). Rat βdefensin-2 ELISA was purchased from Bioassay Technology Laboratory (Shanghai, China). Transforming growth factor-β (TGF-β) ELISA kits were purchased from R&D Systems (Minneapolis, MN). All chemicals were used without further purification. Male SD rats weighing 230-250 g were purchased from Orient (Seoul, Korea). All the procedures and experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Samsung Biomedical Research Institute (SBRI). 2.2. Synthesis of HA-EGF conjugate Aldehyde-modified HA (HA-ALD) was synthesized as described elsewhere.29 Briefly, 1 g of HA with a MW of 200 kDa was dissolved in 100 ml of distilled water. Two molar excess of sodium periodate was added to the HA solution, which stirred for 6 h in the dark. Ethylene glycol (0.5 ml) was added to the reaction mixture and stirred for 2 h to terminate the reaction. The resulting product was poured into a dialysis membrane tube (MWCO = 10 kDa), and dialyzed against 0.1 M NaCl aqueous solution and distilled water for 3 days. Aldehyde content was analyzed by 1H nuclear magnetic resonance (NMR, DPX500, Bruker, Germany). After that, 1 mg of


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EGF dissolved in PBS (pH 7.4) was transferred to sodium acetate buffer (pH 5.5) using Illustra NAPTM-5 columns, which was mixed with HA-ALD having an aldehyde content of 20 mol%. HA-EGF conjugate was synthesized by the coupling reaction between aldehyde groups of HA-ALD and N-terminal amine groups of EGF. The number of EGF molecules per HA chain was determined to be 10, which enabled the bioconjugation efficiency more than 90%.24-26 Sodium cyanoborohydride at 5 molar excess of HA repeating unit was added for the reduction of hydrazine bonds at room temperature for 24 h. After conjugation, 5 molar excess of ethyl carbazate was added with sodium cyanobrohydride and stirred for 24 h to block the residual aldehyde group in HA-EGF conjugates. The resulting HA-EGF conjugate solution was purified using a centrifugal filter (MWCO of 10 kDa) to remove unreacted EGF and other chemicals. The synthesized HA-EGF conjugate was analyzed by GPC comparing the retention time before and after conjugation of HA with EGF. The bioconjugation efficiency of the peptide was calculated from the GPC peak area of unreacted EGF before purification. GPC was performed using the following systems: Waters 717 plus autosampler, Waters 1525 binary HPLC pump, Waters 2487 dual λ absorbance detector, GE Healthcare Superdex Peptide 10/300 GL column. The mobile phase was PBS at pH 7.4 and the flow rate was 0.4 ml/min. The detection wavelength was 280 nm. The concentration of EGF in HAEGF conjugate was determined by Bradford assay. The immunological binding affinity of EGF and HA-EGF conjugate to anti-EGF antibody was assessed by human EGF ELISA. Four replicates were tested for the analysis. 2.3. In vitro serum stability test of HA-EGF conjugates The serum stability of EGF and HA-EGF conjugate was evaluated by ELISA after incubation in human serum at a concentration of 0.1 mg/ml and 37 °C for up to 4 days. At the predetermined time intervals, each sample was immediately diluted by 5,000 times with PBS and stored at - 80 °C before the ELISA. The samples were analyzed by human EGF ELISA kits. Four replicates were performed for the experiment. 2.4. In vitro biological activity test of HA-EGF conjugates 2.4.1. Proliferation of Balb/3T3 cells


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Balb 3T3 cells were cultured in high glucose DMEM supplemented with 10 vol% fetal bovine serum (FBS) and 10 IU/ml of antibiotics. The proliferative activity of EGF and HA-EGF conjugate in skin cells was assessed by MTT assay. The Balb 3T3 cells were suspended at a concentration of 5 × 104 cells/ml in DMEM and 100 µl of the cell suspension containing 5 × 103 cells was seeded on the flat bottom of 96 well cell culture plate. On the next day, the medium was replaced with DMEM supplemented with 0.5 vol% FBS and incubated for 24 h to starve the cells. After 24 h, the medium was replaced with 100 µl of fresh medium in each well and 50 µl of serially diluted EGF and HA-EGF conjugate solutions from 150 ng/ml (3×) at a dilution factor of 5 were treated in quadruplicates for each concentration. The plates were incubated at 37 ºC in a humidified 5% CO2 cell culture incubator for 72 h. Then, MTT reagent was added to each well and the plate was incubated at 37 ºC in the cell culture incubator for 1 h. DMSO (50 µL) was added to each well and the optical absorbance was measured at 540 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). Four replicates were performed for the experiment. 2.4.2. Proliferation of human fibroblasts Normal human dermal fibroblasts were seeded at a low density of 2 × 104 cells/well in a 12-well tissue culture plate and pre-cultured for 24 h before the application of EGF or HA-EGF conjugate at three different concentrations. The controls comprised cultures without proliferation-inducing compounds. Each condition was repeated in triplicate. After incubation for 24 h without change of the culture medium, the cells were washed with PBS twice and the cell proliferation was determined by WST-8 assay using a Cell Counting Kit-8 (CCK-8, Dojindo, Gaithersburg, MD). 2.5. Preparation of HA patch films incorporating HA-EGF conjugates Circular HA patch films were replicated from PDMS molds with 8 mm holes by casting HA solution containing HA-EGF conjugates. The PDMS molds were prepared by curing a mixture of PDMS precursors and curing agents (weight ratio = 10:1) at 60 ºC for 12 h, followed by punching the cured PDMS sheet with an 8 mm biopsy punch. Before casting HA solutions, the molds were sterilized by UV exposure in a clean bench. HA solutions in PBS (pH 7.4, 0.2 ml of 7 w/v%) containing EGF (1 µg) or HA-EGF conjugate (10 µg, EGF 1 µg) were poured into the holes of the PDMS mold, and then dried at 25 ºC for 36 h. The prepared HA patch films (8


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mm disc with a thickness of 0.5 mm) were sealed with a vacuum packing machine and stored at 4 °C prior to use. 2.6. FITC labeling of EGF and HA-EGF conjugate For in vitro release test and fluorescence microscopic imaging, EGF and HA-EGF conjugate were fluorescently labeled with FITC. Twenty molar excess of FITC was added to the solutions of EGF and HA-EGF conjugate dissolved in sodium carbonate buffer (pH 9.0), respectively. The reaction mixture stirred at room temperature in the dark overnight and purified using Illustra NAPTM-5 columns. The degree of labeling modification was assessed by measuring the absorbance at 280 nm and 494 nm. The resulting FITC-labeled EGF (EGF-FITC) or FITC-labeled HA-EGF conjugate (HA-EGF-FITC) solution was incorporated into HA patch films for the following experiments. 2.7. In vitro release tests of HA-EGF conjugates from HA patch films To investigate the release kinetics of HA-EGF conjugate from HA patch films in the hydrate wound area, in vitro static diffusion experiments with FITC-labeled HA-EGF conjugates were performed using the Franz cell.30 EGF-FITC incorporated in HA patch films was used as a control. The Franz diffusion cell with donor and receptor chambers was separated by a 1 wt% agarose hydrogel membrane with an effective area of 1 cm diameter. The receptor chamber with a side arm was filled with 20 ml of fresh PBS buffer (pH 7.4) and maintained at 37 °C. The HA patch films (8 mm disc) containing 1 µg of HA-EGF-FITC or EGF-FITC (based on the amount of EGF), respectively, were positioned onto the agarose membrane in the donor side, and then 1 ml in the receptor chamber was sampled at predetermined time points. After sampling, 1 ml of fresh PBS was refilled to the receptor chamber. The permeation of HA-EGF-FITC (1 µg) in PBS as a control was also assessed after dropping HA-EGF-FITC solution onto the membrane. The amounts of released HA-EGF-FITC or EGFFITC were analyzed by measuring the fluorescence intensity of FITC (GLOMAX®, PROMEGA, Madison, WI). 2.8. Two-photon microscopy after topical delivery of HA-EGF conjugates After anesthetizing SD rats with sodium pentobarbital (100 mg/kg body weight), HA patch films incorporating EGF-FITC or HA-EGF-FITC were topically applied on the hair-removed normal rat skin and


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wound area, respectively. The wound model was prepared by punching the back skin of SD rats with an 8 mm biopsy punch. Animals were sacrificed with a lethal dose of pentobarbital 4 h post-treatment for the skin tissue retrieval. The dissected tissues were fixed in a 4% paraformaldehyde solution. Two-photon fluorescence imaging was carried out with Leica TCS SP5II MP SMD FLIM (Leica, Deerfield, IL). The two-photon fluorescence signals of FITC labeled EGF and HA-EGF conjugate were visualized and the second harmonic generation of collagen structure was obtained using a titanium-sapphire laser at the wavelength of 900 nm. The images were collected as Z-stacks (xyz, 400 Hz) at 512 × 512 pixels and analyzed with LAS AF Lite 2.6.1 of Leica. 2.9. In vivo monitoring of EGF and HA-EGF conjugate in wound tissues In vivo behavior of EGF and HA-EGF conjugate incorporated in HA patch films was monitored by optical imaging and ELISA. For optical imaging, HA patch films incorporating EGF-FITC and HA-EGF FITC with the same fluorescence intensity were topically applied on the wound area. At pre-determined time intervals, the fluorescence signal from EGF-FITC and HA-EGF-FITC treated to the rats was obtained by an optical imaging system (IVIS: Xenogen, Alameda, CA). The photonic emission was assessed with Living Image® 2.2.0.1 Software (Xenogen). The data were presented by pseudocolor representation for light intensity and mean photon in the region of interest (ROI). Two replicates were performed for the in vivo imaging. Animals were sacrificed with a lethal dose of pentobarbital 1 day post-treatment. The dissected skin samples were fixed, embedded in optimal cutting temperature (OCT) compound, and cryosectioned. For ELISA, HA patch films incorporating EGF and HA-EGF conjugate with 1 µg EGF per each patch were topically applied on the wound area. The wound tissues were extracted using a 10 mm biopsy punch at predetermined intervals and the extracted tissues were homogenized. Then, the concentration of EGF was quantified using a human EGF ELISA kit. Three replicates were performed for the experiments. 2.10. In vivo evaluation of HA-EGF conjugates for skin wound healing The wound model was induced using a biopsy punch as described in section 2.8 (n = 5). HA patch film was used as a control group, and HA patch films incorporating EGF and HA-EGF conjugate were treated daily for 8 days. To prevent licking or touching the wound tissues of animals, the wound was induced on the back skin of


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rats and all animals were bred in separated cages. When the patch-type HA film was treated to the wet wound tissue, the film became viscous medium like ointment in the wound environment. As the wound became recovered and the wound size decreased, a drop of normal saline was applied on the wound tissue for the topical attachment of the HA patch-type film. The film became viscous medium and infiltrated into wound tissues. The administered dose of EGF was 1 µg per patch for both EGF and HA-EGF conjugate. On day 2, 4, 6, and 8, wounds were photographed to observe the changes in the wound area. The rats were sacrificed 4 days and 8 days post-treatment for histological analysis and cytokine analysis of wounds. The harvested wound tissues were fixed in 4% formaldehyde at room temperature overnight. The fixed wound tissues were embedded in paraffin, which were cut into serial sections, heated in a vacuum oven at 60 °C for 20 min, deparaffinized, and hydrated. Then, the sections were stained with hematoxylin and eosin (H&E) or Masson’s trichrome. In addition, the homogenate of dissected wound tissues was added to the well of an ELISA kit containing 0.1 ml of the lysis buffer. The cytokine level of the wound tissues was analyzed by ELISA. 2.11. Statistical analysis The data are expressed as means ± standard deviation from several separate experiments. Statistical analysis was carried out via Student’s t-test for the comparison of the two groups, and the one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was performed for the comparison of more than two groups. A value for P < 0.05 was considered statistically significant.


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3. RESULTS AND DISCUSSION 3.1. Synthesis and characterization of HA-EGF conjugates As schematically shown in Fig. 1a, we developed HA-EGF conjugates for skin wound healing. In our previous works24,26, HA appeared to facilitate the transdermal delivery of biomolecules into deep dermal tissues. HA-EGF conjugates can interact with cells in skin tissues such as keratinocytes in epidermis and dermal fibroblasts, and stimulate their growth and differentiation (Fig. 1a). For facile topical application, we incorporated HA-EGF conjugate into a matrix film of high MW HA. The HA patch films containing appropriate amounts of EGF or HA-EGF conjugate for wound healing were prepared by the solvent casting of HA and EGF or HA-EGF conjugate solutions after filling the mixtures into circular cavities of a PDMS mold. The wound model was induced using an 8 mm biopsy punch and the wound healing process was evaluated after treatment with HA-EGF conjugates in the patch-type HA film (Fig. 1b). The patch-type film containing HA-EGF conjugate exhibited the advantages of both general patches and ointment. Before treatment on the wet wound tissue, it has a form of patches for facile topical application and enables to maintain the biological activity of EGF. The patch-type film was prepared without using chemical crosslinkers which might affect the biological activity of EGF. After topical application, it becomes viscous liquid like ointment and delivers HA-EGF conjugates in a sustained manner by infiltrating into wound tissue. HA-EGF conjugate was synthesized and characterized by using several analytical methods. HA with a MW higher than 100 kDa can be used for long-term delivery of peptide or protein drugs without immunogenicity issues of low MW HA. On the other hand, HA fragment shorter than 300 kDa was reported to promote the wound healing.18,19 Thus, HA with a MW of 200 kDa was chosen as a delivery carrier for wound healing. Aldehyde groups were introduced to 200 kDa HA molecules by the treatment with sodium periodate. Then, EGF molecules were evenly grafted to the aldehyde groups on the long chain of HA by reductive amination (Fig. 2a). During the reaction, aldehyde groups on HA can preferably react with N-terminal primary amine groups of EGF rather than ε-amino groups of lysine at a low pH around 5.5 due to their pKa difference.29 The selective conjugation of HA-ALD with the N-terminal amine group of EGF can contribute to maintain the secondary structure and biological activity of EGF after conjugation.


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a S.C. Epidermis

Wound formed by biopsy punch Keratinocytes HA patch incorporating HA-EGF conjugates

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Fibroblasts

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Figure 1. (a) Schematic illustration of hyaluronate-epidermal growth factor (HA-EGF) conjugates incorporated into a patchtype film of HA for skin wound healing. HA-EGF conjugates can penetrate into skin tissues and interact with skin cells via receptors of HA and EGF. (b) Schematic illustration for the preparation of HA patch films incorporating EGF or HA-EGF conjugates for skin wound healing.


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Figure 2. (a) Schematic representation for the chemical structure of hyaluronate-epidermal growth factor (HA-EGF) conjugates. (b) Gel permeation chromatogram (GPC) of EGF and HA-EGF conjugate. (c) The ratios of EGF concentrations in HA-EGF conjugates determined by ELISA and Bradford assay (n = 4). (d) In vitro serum stability of EGF and HA-EGF conjugate in the human serum (n = 3).


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The successful synthesis of HA-EGF conjugates was confirmed by GPC. As shown in Fig. 2b, the peak of HA-EGF conjugate appeared at a shorter retention time than that of EGF or HA, indicating the increase of hydrodynamic size after conjugation reaction between HA-ALD and EGF. From the peak area of unreacted EGF around 27 min before purification, the conjugation efficiency was calculated to be about 90%, which means that 90% of the added EGF was conjugated to HA-ALD. After conjugation, the broad peak of HA-ALD was shifted to that of HA-EGF conjugate around 18 min. The immunological bioactivity of HA-EGF conjugates determined by human EGF ELISA and Bradford assay was comparable to that of native EGF without conjugation (Fig. 2c). The conjugation process appeared not to cause the reduction of immunological bioactivity of EGF, possibly due to the selective conjugation of HA with N-terminal amine groups of EGF. Furthermore, in order to investigate the serum stability of EGF after conjugation to HA, native EGF and HA-EGF conjugate were incubated in human serum at 37 °C for 4 days. According to the analysis by ELISA, EGF was degraded and lost the 40% of immunological activity in human serum within 4 days (Fig. 2d). In contrast, HA-EGF conjugates exhibited excellent serum stability over 4 days. The conjugation of EGF to the long-chain polymer of HA seems to contribute to the protection of EGF from human serum components. 3.2. In vitro biological activity of HA-EGF conjugates

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Figure 3. The proliferative activity of epidermal growth factor (EGF) and hyaluronate-EGF (HA-EGF) conjugate in (a) Balb/3T3 cells (n = 4) and (b) human dermal fibroblasts (n = 6).


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In vitro biological activity of EGF and HA-EGF conjugate was assessed in a mouse embryonic fibroblast cell line of Balb/3T3 cells and human dermal fibroblasts. As shown in Fig. 3, the proliferation of skin cells was monitored with increasing concentration of EGF. The biological activity of HA-EGF conjugates was compared with that of EGF in Balb/3T3 cells using the concentration of 50 % cell growth (ED50). The ED50 value of EGF was 2.79 pg/ml and the value of HA-EGF conjugates was 5.71 pg/ml. The ED50 value was barely changed after conjugation, which indicated that the effect of HA-EGF conjugates on the proliferation of Balb/3T3 cells was comparable with that of native EGF. In addition, both EGF and HA-EGF conjugate showed the remarkable effect on the proliferation of human dermal fibroblasts in a dose-dependent manner. In contrast to the previous report on HA-EGF conjugates synthesized by the non-specific carbodiimide chemistry,31 the biological activity of EGF could be maintained due to the selective conjugation of HA-ALD with N-terminal amine group of EGF, effectively stimulating the skin cell proliferation. Moreover, as reported elsewhere,29,32 long polymer chains can sterically hinder the interaction between drugs and the cells. However, HA-EGF conjugates showed an equivalent biological activity to native EGF, which might be made possible by the mutual interaction of both HA and EGF with their receptors on the cells. 3.3. In vitro release of HA-EGF conjugates from HA patch films For facile topical delivery in a controlled manner during the healing process, HA-EGF conjugate was formulated into a patch-type film of HA. Once the HA patch film contacted on the wet wound, it was immediately hydrated and formed a viscous medium for the diffusion-controlled release of HA-EGF conjugates.33 Thus, the release of HA-EGF conjugates incorporated in the HA patch film could be retarded and controlled depending on the viscosity of the medium and the interaction with the surrounding HA matrix. Because the incorporation of HA-EGF conjugates into the patch-type film of HA by physical interaction was performed by only mild drying process at room temperature, the biological activity of EGF could be maintained by avoiding a harsh process or chemical crosslinking reaction. In vitro release of HA-EGF conjugates from a solution drop and a HA patch film was compared using a static Franz diffusion cell. As shown in Fig. 4, while HA-EGF conjugate in the solution and EGF incorporated in the patch-type HA film were rapidly diffused out


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through the membrane with an initial burst release, the conjugate was relatively slowly released from the patchtype HA film. Since the skin has more dense structures than that of the membrane used for in vitro release tests, it is expected that the sustained release of EGF would become more noticeable and significant in vivo. Considering the wound healing period, sustained and controlled delivery of EGF would be more favorable rather than temporary exposure of EGF to the wound area for effective wound healing.13,14

100

Cumulative release (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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80 60 40 HA-EGF solution Patch-type HA-EGF Patch-type EGF

20 0 0

2

4

6

8

Time (h) Figure 4. In vitro release profiles of hyaluronate-epidermal growth factor (HA-EGF) conjugate from the aqueous solution and the HA patch film assessed by using Franz cells. In vitro release of EGF incorporated in the patch-type HA film was also analyzed as a control (n = 3).

3.4. Two-photon microscopy for topical delivery of HA-EGF conjugates The transdermal delivery of EGF and HA-EGF conjugate incorporated in HA patch films was visualized by two-photon fluorescence microscopy on normal skin and wound area (Fig. 5). The skin tissues were retrieved 4 h after its topical application and the cross-sectional images parallel to the skin surface were collected for comparison. The dermal skin tissue was visualized in blue due to the second harmonic generation (SHG) of collagen.34,35 From the outer layer to the inner layer of dermis, FITC labeled to EGF and HA-EGF conjugate was


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60 µm

120 µm

180 µm

a

EGF

HA-EGF

b Normal EGF Wound

HA-EGF

Figure 5. Two-photon microscopic images of FITC-labeled epidermal growth factor (EGF-FITC) and hyaluronate-EGF (HAEGF-FITC) conjugate incorporated in HA patch films 4 h after treatment on (a) normal skin tissues and (b) wound tissues. Because the wound area is a void by removing skin tissues with a biopsy punch, nothing was detected. The images are the cross-sections of deep skin tissues 60, 120, 180 µm apart from the skin surface, respectively (scale bars = 250 µm).


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visualized to evaluate their penetration into skin tissues. As shown in Fig. 5a, HA-EGF conjugates penetrated into deep skin tissues over the wide dermal region more readily than EGF. In contrast, EGF was only observed in the follicular area. These results might be attributed to the characteristics of HA with a hydrophobic patch domain and its interaction with lipid components of stratum corneum, the outermost skin barrier.22,23 Furthermore, HA receptors and EGF receptors on keratinocytes and fibroblasts might facilitate the transdermal delivery of HA-EGF conjugates. The transdermal delivery of EGF and HA-EGF conjugate was also investigated in wound tissues. In this case, skin tissues including stratum corneum, epidermis and dermis were removed with the biopsy punch. The penetration of EGF and HA-EGF conjugate into the surrounding normal tissues was observed in the boundary region between normal skin tissues and wound area. As shown in Fig. 5b, there was no obvious signal in the wound area, whereas green signal of FITC labeled to samples and blue signal of SHG from the collagen of dermal skin tissues could be observed in the surrounding normal tissues. The strong FITC signal of HA-EGF conjugates might reflect the penetration of the conjugates into the peripheral tissues via the interaction with HA and EGF receptors on the skin cells. In contrast, FITC signal was rarely observed for EGF samples. In wound tissues, proteases are reported to be upregulated1 and the rapid degradation of EGF is considered as the drawback of EGF therapy for wound healing.36 In other words, EGF might be degraded in wound area rather than absorbed into the peripheral tissues. The EGF in HA-EGF conjugates might be protected from the proteases by the shielding effect of HA.37 The EGF delivered into the peripheral tissues might stimulate proliferation, differentiation and migration of the skin cells, resulting in effective wound healing. 3.5. In vivo monitoring of EGF and HA-EGF conjugate in wound tissues The transdermal delivery of EGF and HA-EGF conjugate was investigated by optical imaging and ELISA. The fluorescence signal of FITC-labeled EGF and HA-EGF conjugate incorporated in HA patch films was monitored over 16 h. As shown in Fig. 6a and 6b, the fluorescence signal of EGF-FITC disappeared in 4 h, but the signal of HA-EGF-FITC remained for more than 8 h. After rapid degradation of EGF in wound tissues, FITC might be released from EGF and diffused out to the surrounding tissues due to its small size, whereas HA-EGFFITC resulted in sustained degradation of EGF due to the steric hindrance of HA. To make it clearer, ELISA was


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a

1h

4h

8h

16 h

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HA-EGF

c

b

100

5 EGF HA-EGF

4 3 2

EGF HA-EGF

80

EGF (pg/ml)

Relative fluorescence (x109 p/sec/cm 2/sr)

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1

**

60

**

40 20

0

0 0

1

4

8

16

Time after treatment (h)

1

4

8

Time after treatment (h)

d

Figure 6. (a) Fluorescence images for the degradation behavior of epidermal growth factor (EGF) and hyaluronate-EGF (HAEGF) conjugate in patch-type HA films after topical application of FITC-labeled samples on wound tissues. The transdermal delivery was quantitatively analyzed by (b) fluorescence signal and (c) ELISA (n = 3, ** P < 0.01 versus EGF-treated group). (d) The crosssectional fluorescence microscopic images of the skin tissues excised a day post-treatment of FITC-labeled (left) EGF and (right) HA-EGF conjugate (scale bar = 200 µm).


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performed using the homogenate of excised skin tissues (Fig. 6c). HA-EGF conjugate showed a longer residence time in wound area due to the HA chain preventing the degradation by proteases and facilitating the cellular uptake of EGF into skin cells via the interaction with HA receptors. The cross-sectional images a day after treatment with EGF-FITC and HA-EGF-FITC showed that HA-EGF conjugate still remained in the peripheral skin tissues near the wound area (Fig. 6d). As reported elsewhere8, many proteases easily decomposed EGF in skin wound sites. The long-lived species of EGF-EGFR complex on the cell surface is highly required for effective wound healing. Thus, the HA-EGF conjugate with a prolonged residence time might greatly contribute to the facilitated skin wound healing. 3.6. In vivo assessment of HA-EGF conjugates incorporated into HA patch films The wound healing effect of HA-EGF conjugates incorporated into HA patch films was assessed in the dorsal skin of SD rats. When the model wound was prepared on skin tissues using an 8 mm biopsy punch, the wound area was hydrated within 5-10 min by the wound fluid effused from the scar (Fig. 1b). HA patch films appeared to be swollen and dissolved into the viscous liquid state. The dissolved HA patch film could be steadily attached to the wound area. As shown in Fig. 4, HA-EGF conjugates might be slowly diffused from the viscous gel-like solution. To measure the wound size, the wound area was photographed every 2 days (Fig. 7a). As shown in Fig. 7b, the wound area measured each day was normalized by regarding the initial wound area as 100%. The half healing time (HT50), the time for the reduction of wound area to half of the initial size, was calculated by extrapolating from the measured data. Although the wound size decreased by natural healing for the untreated control group, the wound closure process was more accelerated for groups treated with EGF and HA-EGF conjugate in the patch-type HA films. The HT50 for the case of HA-EGF conjugate was only 3.11 days, which was greatly lower than 5.50 days for the control group. Remarkably, it was also statistically lower than 3.92 days of EGF in a patch-type HA film, reflecting the therapeutic effect of HA-EGF conjugates on skin wound healing. Histological analysis with H&E staining and Masson’s trichrome staining was performed for further therapeutic analysis (Fig. 8). On the punched skin tissues, damaged skin tissues showed loosely arranged and less granulated tissue structures as reported elsewhere.38 Although wound closure was almost completed in epidermal tissues


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a

Day 0

Day 2

Day 4

Day 6

Day 8

Control

HA

EGF

HA-EGF

b

100 HT50 80

Wound area (%)

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60

Control HA

5.50 5.35

EGF

3.92

HA-EGF

3.11

**

40

* 20

*** ***

0 0

2

4

6

8

Time (days) Figure 7. (a) Photographic images of wound areas for 8 days and (b) the changes in measured wound area after treatment with HA patch films, HA patch films incorporating epidermal growth factor (EGF) and hyaluronate-EGF (HA-EGF) conjugate (n = 5). The graph is extrapolated by nonlinear regression and the calculated half healing times (HT50) are represented in the legend box (*P < 0.05, ** P < 0.01 and ***P < 0.001 versus EGF-treated group).


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Masson’s trichrome

Normal

Control

HA

EGF

HA-EGF

Figure 8. Histological analysis with H&E staining and Masson’s trichrome staining 8 days after treatment with HA patch films, HA patch films incorporating epidermal growth factor (EGF) and hyaluronate-EGF (HA-EGF) conjugate on wound model tissues (scale bars = 500 µm).


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after 8 days, the damaged dermal tissue area was still large for the untreated control and the HA patch film treated group. However, after treatment with HA-EGF conjugates in HA patch films, damaged area was significantly reduced and the border between damaged tissues and normal tissues was ambiguous compared with other groups. In addition, the recovered skin tissues showed almost complete re-epithelialization and granulation of dermal tissues. Particularly, HA-EGF conjugate treated group showed a fully recovered hypodermis structure with white granular tissues, which might be attributed to the facilitated penetration of HA-EGF conjugates into deep dermal tissues and the prolonged residence of HA-EGF conjugate. Masson’s trichrome staining also showed the positive effect of HA-EGF conjugate on the skin wound healing in consistent with the results of H&E staining. The EGF treated group and especially HA-EGF conjugate treated group showed a large area deposition of collagen. It was reported that the formation of granular tissue resulted from the enhanced accumulation of collagen and glycosaminoglycan during the wound healing.39,40 The cytokine analysis in skin tissues also supported the therapeutic effect of HA-EGF conjugates in a patchtype HA film (Fig. 9). HA-EGF conjugates reduced the high level of inflammatory cytokines such as TNF-α and IL-1 induced by wound generation more effectively than other groups including EGF-treated group. Interestingly, three groups treated with HA patch films showed the increased level of β-defensin 2. It was reported that HA might induce the activation of keratinocytes by producing β-defensin 2.18 Thus, HA patch films might contribute to the wound healing as well as working as a facile sustained delivery platform. HA-EGF conjugates in HA patch films also significantly elevated the level of TGF-β known to stimulate the cellular proliferation. Taken together, we could confirm the feasibility of HA-EGF conjugates in a patch-type film of HA from the excellent therapeutic effect on skin wound healing due to the long-term release of the conjugate from the HA patch film, its improved penetration into skin tissues, the prolonged residence of EGF in wound tissues and the skin self-defense system induced by HA.


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b

a

120 100

150 100

**

IL-1 (pg/ml)

TNF-α α (pg/ml)

****

****

200

50

80 60 ** 40 20

0

ol al rm n tr o o N C

0 HA

E

GF

GF -E A H

c

al ol rm n tr o o N C

HA

F F EG A-EG H

d 40 50

***

**

TGF-β β (pg/ml)

β -defensin-2 (pg/ml)

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40 30 20

30

*

20

10

10 0

al ol rm n tr No Co

0 HA

F F EG A-EG H

al ol rm n tr No Co

HA

F F EG A-EG H

Figure 9. Cytokine analysis for (a) TNF-α, (b) IL-1, (c) β-defensin-2 and (d) TGF-β in the homogenate of dissected wound tissues 8 days after treatment with HA patch films, HA patch films incorporating epidermal growth factor (EGF) and hyaluronate-EGF (HA-EGF) conjugate on wound model tissues (n = 4, *P < 0.05, **P < 0.01 and ***P < 0.001).

4. CONCLUSIONS We developed HA-EGF conjugates incorporated into a patch-type film of HA for skin wound healing by facilitated delivery and prolonged residence of EGF in wound tissues. First, we synthesized HA-EGF conjugates by conjugating amine group of EGF to aldehyde-modified HA by reductive amination. Then, we confirmed in vitro antibody-binding activity, improved serum stability and biological activity of HA-EGF conjugates for skin cell proliferation. As the next step, we incorporated HA-EGF conjugates into HA patch films by the drying


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method at room temperature for facile application onto the skin wound. With the sustained release from the patch-type film, HA-EGF conjugates efficiently penetrated into peripheral skin tissues around the wound area with a long residence time against the protease in the wound tissues. HA-EGF conjugates in HA patch films showed great therapeutic effect on wound healing compared with other control groups. Taken together, we could confirm the feasibility of HA-EGF conjugate for skin wound healing and other skin related diseases.

AUTHOR INFORMATION * Corresponding Author Tel.: +82 54 279 2159; Fax: +82 54 279 2399; E-mail address: [email protected] (S. K. Hahn). Tel.: +82 55 350 5382; Fax: +82 55 350 5389; E-mail address: [email protected] (S. Y. Yang). Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources The authors declare no competing financial interest.

ACKNOWLEDGMENTS This research was supported by the Bio & Medical Technology Development Program (No. 2012M3A9C6049791), Mid-career Researcher Program (No. 2015R1A2A1A15053779) and Basic Science Research Program (NRF2013R1A1A1075975) of the National Research Foundation (NRF) funded by the Ministry of Science, ICT & Future Planning, Korea.


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31. Ferguson, E.L.; Alshame, A.M.J.; Thomas, D.W. Evaluation of hyaluronic acid-protein conjugates for polymer masked-unmasked protein therapy. Int. J. Pharmaceut. 2010, 402, 95-102. 32. Yang, J. –A.; Kong, W. H.; Sung, D. K.; Kim, H.; Kim, T. H.; Lee, K. C.; Hahn, S. K. Hyaluronic AcidTumor Necrosis Factor-Related Apoptosis-Inducing Ligand Conjugate for Targeted Treatment of Liver Fibrosis. Acta Biomater. 2015, 12, 174-182. 33. Puttipipatkhachorn, S.; Nunthanid, J.; Yamamoto, K.; Peck, G. E. Drug Physical State and Drug-Polymer Interaction on Drug Release from Chitosan Matrix Films. J. Controlled Release 2001, 75, 143-153. 34. Williams, R. M.; Zipfel, W. R.; Webb, W. W. Interpreting Second-Harmonic Generation Images of Collagen I Fibrils. Biophys. J. 2005, 88, 1377-1386. 35. Zipfel, W. R.; Williams, R. M.; Christie, R.; Nikitin, A. Y.; Hyman, B. T.; Webb, W. W. Live Tissue Intrinsic Emission Microscopy Using Multiphoton-Excited Native Fluorescence and Second Harmonic Generation. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 7075-7080. 36. Okumura, K.; Kiyohara, Y.; Komada, F.; Iwakawa, S.; Hirai, M.; Fuwa, T. Improvement in Wound Healing by Epidermal Growth Factor (EGF) Ointment. I. Effect of Nafamostat, Gabexate, or Gelatin on Stabilization and Efficacy of EGF. Pharm. Res. 1990, 7, 1289-1293. 37. Oh, E. J.; Park, K.; Kim, K. S.; Kim, J.; Yang, J. –A.; Kong, J. –H.; Lee, M. Y.; Hoffman, A. S.; Hahn, S. K. Target Specific and Long-Acting Delivery of Protein, Peptide, and Nucleotide Therapeutics Using Hyaluronic Acid Derivatives. J. Controlled Release 2010, 141, 2-12. 38. Braiman-Wiksman, L.; Solomonik, I.; Spira, R.; Tennenbaum, T. Novel Insights into Wound Healing Sequence of Events. Toxicol. Pathol. 2007, 35, 767-779. 39. Laato, M.; Ninikoski, J.; Lebel, L.; Gerdin, B. Stimulation of Wound Healing by Epidermal Growth Factor. Ann. Surg. 1986, 203, 379-381. 40. Hennessey, P. J.; Black, C. T.; Andrassy, R. J. EGF Increases Short-Term Type I Collagen Accumulation During Wound Healing in Diabetic Rats. J. Pediatr. Surg. 1990, 25, 893-897.


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Biomacromolecules

TOC FIGURE

S.C. Wound formed by biopsy punch

Epidermis Dermis

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Keratinocytes HA patch incorporating HA-EGF conjugates

Fibroblasts

HA receptor

EGF receptor

HA-EGF conjugates


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HyaluronateâEpidermal Growth Factor Conjugate for Skin Wound

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