Elsevier

Biomaterials

Volume 33, Issue 25, September 2012, Pages 5947-5954
Biomaterials

Transdermal delivery of hyaluronic acid – Human growth hormone conjugate

https://doi.org/10.1016/j.biomaterials.2012.05.003Get rights and content

Abstract

Hyaluronic acid (HA) is one of the major components of extracellular matrix (ECM). Keratinocyte and fibroblast are known to have HA receptors in the skin. Fibroblast also has human growth hormone (hGH) receptors. In this work, HA–hGH conjugate was developed as a receptor mediated transdermal delivery system of protein drugs. HA–hGH conjugate was synthesized by specific coupling reaction between aldehyde modified HA and the N-terminal amine group of hGH. We could confirm the proliferative effect of HA on keratinocyte and fibroblast, and the biological activity of HA–hGH conjugate in fibroblast with an elevated expression level of phosphorylated Janus kinase 2 (p-JAK2). Interestingly, fluorescence microscopy clearly visualized the dramatically enhanced penetration of HA–hGH conjugate through the dorsal skin of mice after topical treatment with FITC labeled HA–hGH conjugate. According to pharmacokinetic analysis, HA–hGH conjugate appeared to be delivered through the skin into the blood stream possibly by the receptor mediated transdermal delivery. This work confirms the feasibility of using the HA–hGH conjugate as a model system for the receptor mediated transdermal delivery of protein drugs and their further exploitation for various cosmetic and tissue engineering applications.

Introduction

Recently, a number of transdermal drug delivery systems have been developed mainly due to their advantages such as effective systemic delivery bypassing digestive systems, patient compliance without painful injections, and easy control to terminate drug delivery [1], [2], [3]. Despite these benefits, a low bioavailability is one of the major disadvantages of transdermal drug delivery systems due to the poor skin permeability. The outermost layer of epidermis, stratum corneum, is the inevitable barrier consisting of highly ordered dead cells with intercellular lipids [4]. To circumvent this limitation, various methods have been developed using microneedle patch [5], [6], iontophoresis [7], [8], penetration enhancer [9], [10], and ultrasound [11], [12]. Microneedle patches employ an array of needles in a micron scale to create holes in stratum corneum for skin permeability enhancement. However, it requires multiple and complicate processes to develop microneedle arrays containing drugs, especially for the case of protein drugs [5], [6]. Iontophoresis uses a continuous low voltage current to provide an electrical driving force for charged molecules and an electroosmotic flow for uncharged molecules to deliver drugs through stratum corneum without pain [7], [8]. However, the application of iontophoresis has been limited mostly to the delivery of small molecules with a charge. Ultrasound disrupts the lipid structure of stratum corneum to enhance the skin permeability, which may cause deep tissue damages in some cases [11], [12]. Currently, there are few reports on the efficient transdermal delivery systems of protein drugs.

Hyaluronic acid (HA) is a linear polysaccharide in the body. More than 50% of HA is present in the skin tissue [13], [14], [15]. Despite the high molecular weight and hydrophilicity of HA, it is known to be delivered through the skin tissue in both mouse and human [16], [17]. The mechanism for transdermal transport of HA has not been clearly verified yet, but there are some possible reasons for the positive effect of HA on transdermal delivery. First, HA is very hygroscopic and can hydrate the stratum corneum enhancing the permeability of skin. Second, the hydrophobic patch domain in HA chain can enhance the permeability of HA across the stratum corneum. Third, HA receptors distributed in the skin tissue may facilitate the localization of HA in the skin tissue [17], [18], [19]. Moreover, it is reported that HA can induce the proliferation, migration, adhesion, and differentiation of keratinocyte [20], [21], [22], [23]. HA can also enhance the proliferation of fibroblast through CD44 receptors on the cell membrane [24]. Meanwhile, human growth hormone (hGH) has been widely used for the treatment of short stature by daily injection for months to years. It is well known that hGH receptors are distributed in the skin tissue and have important roles for cell proliferation, mitosis, and differentiation [25], [26], [27]. Especially, hGH promotes the synthesis of insulin-like growth factor I (IGF-1) in fibroblast [25] and the released IGF-1 can also enhance the proliferation of keratinocyte [26].

In this work, on the basis of possitive effect of HA on the transdermal delivery, HA–hGH conjugate was developed as a receptor mediated transdermal delivery system of protein drugs. HA–hGH conjugate was synthesized by coupling reaction of aldehyde modified HA (HA-ALD) with N-terminal primary amine group of hGH. The resulting HA–hGH conjugate was characterized by gel permeation chromatography (GPC) and circular dichroism (CD) spectroscopy. After confirmation of in vitro biological activity of hGH conjugated to HA from the elevated expression level of phosphorylated Janus kinase 2 (p-JAK2) in the fibroblast of Detroit 551 cell, the effect of HA and HA–hGH conjugate was investigated on the proliferatation of human keratinocyte and fibroblast. Then, in vivo skin penetration of HA–hGH conjugate was visualized by fluorescence microscopy after topical treatment of FITC labeled HA–hGH conjugate. Finally, pharmacokinetic (PK) analysis of topically delivered HA–hGH conjugate was carried out to confirm the feasibility of HA–hGH conjugate as a model system for the receptor mediated transdermal delivery of protein drugs with the discussion for their further exploitation for various cosmetic and tissue engineering applications.

Section snippets

Materials

Sodium hyaluronate, the sodium salt of hyaluronic acid (HA), with a molecular weight of 100 kDa was obtained from Shiseido (Tokyo, Japan). Human growth hormone (hGH) was kindly provided by LG Lifesciences (Daejeon, Korea). Human serum, sodium periodate, sodium cyanoborohydride, ethyl carbazate, and tert-butyl carbazate were purchased from Sigma–Aldrich (St. Louis, MO). Human epidermal keratinocytes – neonatal (HEKn), EpiLife medium, fetal bovine serum (FBS), and phosphate buffered saline (PBS)

Synthesis and characterization of HA–hGH conjugate

Fig. 1 shows the schematic representations for the chemical structure of HA–hGH conjugate and the possible mechanism for its receptor mediated transdermal delivery in the skin tissue. It has been previously reported that the receptors of HA and hGH are distributed in the skin tissue, and HA can penetrate even to the dermis [16]. The HA–hGH conjugate binds to HA and hGH receptors on keratinocytes in the epidermis and fibroblasts in the dermis promoting cell proliferation and the synthesis of

Conclusions

HA–hGH conjugate was successfully synthesized by the coupling reaction of HA–ALD with N-terminal primary amine group of hGH for receptor mediated transdermal delivery. GPC analysis confirmed the successful synthesis of HA–hGH conjugate. The number of hGH molecules in HA–hGH conjugate could be controlled in the range from 1 to 9 by changing the amount of hGH in the feed with a bioconjugation efficiency higher than 95%. CD analysis revealed the maintenance of the secondary structure of hGH even

Acknowledgments

This research was supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0081871 and 2011K000801). This study was also supported by a grant of the Korea Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A080711). This work was supported by a grant from the Next-Generation BioGreen 21 Program (No. PJ007974), Rural Development Administration,

References (30)

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These authors contributed equally to this work.

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