Physicochemical properties of pH-sensitive hydrogels based on hydroxyethyl cellulose–hyaluronic acid and for applications as transdermal delivery systems for skin lesions

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Contents lists available at ScienceDirect

European Journal of Pharmaceutics and Biopharmaceutics journal homepage: www.elsevier.com/locate/ejpb

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Research Paper

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Physicochemical properties of pH-sensitive hydrogels based on hydroxyethyl cellulose–hyaluronic acid and for applications as transdermal delivery systems for skin lesions

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Soon Sik Kwon, Bong Ju Kong, Soo Nam Park ⇑

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Department of Fine Chemistry, Seoul National University of Science and Technology, 232 Gongreung-ro, Nowon-gu, Seoul 139-743, South Korea

a r t i c l e

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Article history: Received 19 December 2014 Revised 16 February 2015 Accepted in revised form 26 February 2015 Available online xxxx Keywords: pH-sensitive Hydrogel Smart hydrogel Hyaluronic acid Antimicrobial activity Transdermal delivery system Isoliquiritigenin Propionibacterium acnes

a b s t r a c t We investigated the physicochemical properties of pH-sensitive hydroxyethyl cellulose (HEC)/hyaluronic acid (HA) complex hydrogels containing isoliquiritigenin (ILTG), and discuss potential applications as transdermal delivery systems for the treatment of skin lesions caused by pH imbalance. HA has favorable skin compatibility and pH and HEC serves as scaffold to build hydrogels with varied HCE:HA mass ratio. Hydrogels were synthesized via chemical cross-linking, and three-dimensional network structures were characterized via Fourier transform infrared (FT-IR) spectroscopy. The swelling properties and polymer ratios of the hydrogels were investigated at pH values in the range 1–13. HECHA13 (i.e., an HEC:HA mass ratio of 1:3) was found to have optimal rheological and adhesive properties, and was used to investigate the drug release efficiency as a function of pH; the efficiency was greater than 70% at pH 7. Antimicrobial activity assays against Propionibacterium acnes were conducted to take advantage of the pH-sensitive properties of HECHA13. At pH 7, we found that HECHA13, which contained ILTG, inhibited the growth of P. acnes. Furthermore, HECHA13 was found to exhibit excellent permeability of ILTG into the skin, which penetrated mostly via the hair follicle. These results indicate that this pH-sensitive hydrogel is effective as a transdermal delivery system for antimicrobial therapeutics, with potential applications in the treatment of acne. Ó 2015 Published by Elsevier B.V.

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1. Introduction

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Sensitivity to pH is an important property for transdermal delivery systems. The pH of the skin surface, or stratum corneum (SC), is typically in the range 5.0–6.0. This is termed the ‘acid mantle’, and the pH is influenced by number of internal and external factors, including epidermal cells, glands (sebaceous, apocrine, and eccrine), age, and gender. Maintaining the pH of the SC in the range 5.0–6.0 is important in providing an effective barrier, as well as maintaining physiological processes, due to the optimal structure of the intercellular lipid and SC homeostasis [1,2]. SC intercellular lipids form a stabilized double lamellar structure in these mildly acidic conditions, whereas micellization occurs for pH >6.0 and a disordered structure occurs for pH <4.5 [3]. This unbalanced pH (break down the acid mantle) decreased the SC

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⇑ Corresponding author at: Nanobiocosmetic Laboratory, Department of Fine Chemistry, College of Nature and Life Science, Seoul National University of Science and Technology, 232 Gongreung-ro, Nowon-gu, Seoul 139-743, South Korea. Tel.: +82 2 970 6451; fax: +82 2 972 9585. E-mail address: [email protected] (S.N. Park).

cohesion and permeability barrier, and also skin illness occurs such as inflammation, acne and irritant contact dermatitis [1]. Acne is the common form of skin lesion, and is particularly common among teenagers, although it may present at any age [4]. Hydrogels are typically composed of a natural polymer, such as hyaluronic acid (HA), collagen, gelatin, or cellulose, and are threedimensional networks of hydrophilic polymer chains. Swollen hydrogels absorb large quantities of water without dissolution. Furthermore, hydrogels feature unique mechanical and structural properties, which are similar to those of tissues and to the extracellular matrix (ECM) of the skin. The unique properties of hydrogels, make them suitable for applications as biomaterials for scaffolds in the tissue engineering, drug delivery systems, imaging, therapeutics, and medical devices [5–7]. To exploit the potential of these materials, novel smart hydrogels have been developed by using environmental stimuli-responsive polymers that are designed to respond to environmental factors such as pH, temperature, enzymes, and electric or magnetic fields [8–10]. Smart hydrogels are composed of functional groups on the polymer backbone of the structure, which result from noncovalent bonding, such as hydrogen bonding, hydrophobic interactions, p–p stacking, or

http://dx.doi.org/10.1016/j.ejpb.2015.02.025 0939-6411/Ó 2015 Published by Elsevier B.V.

Please cite this article in press as: S.S. Kwon et al., Physicochemical properties of pH-sensitive hydrogels based on hydroxyethyl cellulose–hyaluronic acid and for applications as transdermal delivery systems for skin lesions, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.02.025

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electrostatic interactions. When smart hydrogels are exposed to environmental factors, the porosity and hydrophilicity of the hydrogel can regulate the loading and release of drug in a controlled manner. Because of this self-regulating behavior, smart hydrogels are promising candidate materials for drug delivery systems [11–13]. HA is a linear polysaccharide composed of repeating units of b-1,4-linked N-acetyl-D-glucosamine and glucuronic acid. The pKa of the carboxyl groups of the HA is in the range 3–4, and these groups are ionized at pH 7. HA is important in skin, and is involved in regulating cell proliferation, migration, and differentiation in the epidermis. It is a primary constituent of the ECM and controls tissue physiological function in the dermis. Furthermore, HA is capable of binding to peptides, matrix proteins, and growth factors [14–17]. HA is a hydrophilic polymer that can absorb large quantities of water and can contain up to 1000 fold more water than its solid volume, due to formation of the hydrogen bonding between carboxyl, and N-acetyl groups of HA with water. The favorable biocompatibility, lack of toxicity, and biodegradation properties of HA make it suitable for a wide range of medical applications, including ocular medicine, plastic surgery, tissue engineering, and drug delivery. HA is commonly used as an ingredient in cosmetics, provides anti-aging and moisture supplying effects to the skin, and is often used as fillers during cosmetic surgery to fill wrinkles and grooves caused by facial aging [18–21]. Cellulose is a common, naturally occurring polymer of glucose; however, it is insoluble in water, as well as many other organic solvents. Cellulose-based derivatives have been developed to improve the solubility of cellulose, including methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), ethyl cellulose (EC), hydroxyethyl cellulose (HEC), and sodium carboxymethylcellulose (NaCMC) [22]. Isoliquiritigenin (ILTG) is a component of Glycyrrhiza uralensis, and is distributed in China and other Southeast Asian countries as a traditional medicine. There have been many reports of beneficial effects of ILTG, including inhibition of the growth of prostate cancer, reduction of prostaglandin E2 and nitric oxide, and inhibition of tyrosinase [23–26]. ILTG also exhibits antimicrobial activities, which are superior to those of other licorice ingredients (glycyrrhizin, liquiritin, liquiritigenin), against Bacillus subtilis, Propionibacterium acnes, and Pseudomonas aeruginosa. Furthermore, the ethyl acetate fraction of Korean licorice contains more ILTG compared with licorice from other countries, including China and Uzbekistan [27]. In this study, we assessed the physicochemical properties of hydroxyethyl cellulose–hyaluronic acid (HECHA) complex hydrogels containing ILTG, and investigated their suitability for applications in transdermal delivery. Hydrogels were prepared with various HEC:HA mass ratios, and the physicochemical properties were investigated using Fourier transform infrared (FT-IR) spectroscopy, scanning electron microscopy (SEM), thermogravimetric analysis (TGA), swelling ratio and rheological characterization, as well as texture analysis. Potential applications in transdermal delivery system were investigated by considering drug incorporation and release efficiency, antimicrobial activity, and permeation profile, which was characterized using confocal laser scanning microscope (CLSM).

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2. Materials and methods

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2.1. Materials

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HA was kindly provided by Bioland (Cheonan, Korea). The molecular weight (Mw) of HA is approximately 0.8 MDa. HEC (average Mw = 90,000 Da), divinyl sulfone (DVS), Nile red (BioReagent, suitable for fluorescence, P98.0%), sodium chloride (NaCl), sodium phosphate dibasic dodecahydrate (Na2HPO412H2O), and ILTG

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were purchased from Sigma Aldrich (St. Louis, MO, USA). Sodium hydroxide (NaOH, assay = 98.0%) and hydrochloric acid (HCl, assay = 35.0%) were purchased from OCI Co. Ltd. (Seoul, Korea). Sodium dihydrogen phosphate dihydrate was purchased from Junsei Chemical Co. Ltd. (Tokyo, Japan). 1,3-Butylene glycol (1,3BG) and ethanol were used as received without further purification. P. acnes ATCC6919 was provided by the Korean Culture Center of Microorganisms (KCCM, Seoul, Korea). A differential reinforced clostridial agar and medium were purchased from Becton, Dickinson and Company (BD, Franklin Lakes, NJ, USA). Water was deionized and Milli-Q filtered.

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2.2. Preparation of HECHA hydrogels

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HECHA complex hydrogels were prepared by changing the weight ratio of HEC to HA by 100:0, 75:25, 50:50, 25:75, and 0:100 wt%, which were coded as HECHA10, HECHA31, HECHA11, HECHA13, and HECHA01 hydrogels, respectively. After measuring the weights of HEC and HA powders, they were dissolved completely in 10 mL 0.02 M NaOH solution by stirring (rpm: 250, for 1.5 h at room temperature). Then, the DVS cross-linker was added into the solution with a 1:1 M ratio of DVS to the repeating unit of HA. The use of DVS as the hydrogel chemical cross-linker showed biocompatibility in vitro and in vivo [28]. After 12 h incubation, the cross-linked hydrogel was washed repeatedly with distilled water to remove unreacted cross-linker and residue and to neutralize the pH [29]. For further experiments, hydrogels were cut into approximately 1 cm  1 cm  0.5 cm (width, length, height) and then freeze-dried for 4 days by using a lyophilizer (60 °C, 5 mmTorr, 4 days; Ilshin biobase, Seoul, Korea).

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2.3. Characterization of HECHA hydrogels

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The composition of HEC, HA, and dried hydrogels was characterized by FT-IR (Nexus Nicolet FTIR, Thermo Scientific, Idstein, Germany). The spectra were recorded in the range of 500– 4000 cm1, and 32 scans were implemented by using a Smart Orbit ATR accessory with diamond crystal and Omnic 8.0 software. The surface morphology of the dried hydrogel was conducted by using SEM (TESCAN VEGA3, Cranberry TWP, PA, USA) with 20 kV accelerating voltage. Cross-sections of dried hydrogel were prepared by using a sharp razor blade. The internal structure (crosssection) was sputtered with gold, then observed, and imaged. Thermogravimetric analysis of HECHA hydrogels was carried out on a JP/DTG-60 (Shimadzu, Tokyo, Japan) in an air-conditioned environment with a flow rate of 60 mL/min, at a heating rate of 10 °C/min in the temperature range of 25–250 °C (initial sample weight: 7.4 mg). The standard material used was a-alumina powder.

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2.4. Swelling ratio of HECHA hydrogels

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The swelling ratio (SR) of HECHA hydrogels was measured by changing the pH at a constant temperature of 37 °C, to simulate the body temperature and application of bio-medicals. The dried hydrogel (Hd, established initial weight of hydrogel: 0.02 g) was immersed in pH solutions with different pH, which was regulated by using HCl and NaOH solutions, ranging from 1 to 13. pH of the solutions was determined by using a pH meter (Mettler Toledo, Seven Compact, Gießen, Germany). After the equilibration time (24 h), swelled hydrogels were taken out, excess solvent on the surface was gently removed, and then the swelled hydrogels (Hw) were weighed. The following Eq. (1) was used to calculate the swelling ratio of the hydrogel:

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SR ð%Þ ¼ ðHw  Hd Þ=Hw  100;

ð1Þ

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where Hw is the weight (g) of the swelled hydrogel, and Hd is the weight (g) of the dried hydrogel.

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2.5. Rheological and mechanical properties of HECHA hydrogels

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The rheological properties of the HECHA hydrogels were investigated using a TA HR-1 rheometer (TA instruments, Leatherhead, UK), which had a 40 mm diameter parallel plate geometry. All samples were loaded carefully onto a peltier plate to minimize shearing, and rheology studies were performed at 25 °C. To investigate the linear viscoelastic regime within 10% [30], oscillatory strain sweep tests were used, in which the hydrogel was deformed at different shear strains; we used a frequency of 1 Hz and the maximum strain was 1%. The maximum strain was held at 1%. The dynamic shear storage modulus G0 and loss modulus G0 0 of HECHA hydrogels were characterized at angular frequencies in the range 0.1–10 rad/s. A texture analyzer (TA.XT Express Enhanced, Stable Micro Systems, Godalming, Surrey, UK) equipped with a 5-kg load cell was used to measure hardness, cohesiveness, and adhesiveness. A 40-mm disk was compressed into the hydrogel and redrawn, where the strain was 30%. A pre-, post- and test speeds were each 2.00 mm/s. The probe was situated above the surface of the gel.

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2.6. Drug incorporation and release efficiency

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To assess the drug incorporation efficiency of the hydrogel, dried hydrogel samples with an established initial mass of 0.02 g, were allowed to swell in 3 mL 20% 1,3-butylene glycol (1,3-BG) solution containing 500 lM ILTG at 37 °C for 12 h in a darkroom (ILTG is light-sensitive). This study was conducted in the darkroom at 37 °C for 12 h because ILTG is sensitive to light. The incorporation efficiency was analyzed using high-performance liquid chromatography (HPLC) (Shimadzu, Tokyo, Japan) with an ILTG calibration curve. The HPLC equipment was comprised of an LC20AT pump, SPE-M20A ultraviolet–visible detector, and a Shippack (VP-ODS) C18 analytical column (5 lm, 25094.6 mm). The mobile phase was a mixture of 2% (v/v) acetic acid in distilled water and 0.5% (v/v) acetic acid in 50% (v/v) acetonitrile. The detection wavelength was 372 nm, and the flow rate was 1.0 mL/min. The following expression was used to calculate the concentration of ILTG that incorporated into hydrogel matrix:

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After incubation of P. acnes, 50 lL of medium was cultured on a clostridial agar plate at 37 °C for 48 h. The antimicrobial activity was measured by confirming the degree of colony size and number of colonies on the plate.

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2.8. In vitro skin permeation

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To investigate their potential as a transdermal delivery system, the HECHA hydrogels were allowed to swell in 0.02% Nile red solution before imaging using CLSM (LSM 5 Exciter, Carl Zeiss, Jena, Germany). Images were obtained in differential interference contrast (DIC) mode, fluorescence mode, and merge mode. The CLSM images were characterized using LSM Image Browser software, version 4.2.0.121 (Carl Zeiss Microimaging, Gottingen, GMBH). Dorsal skin was obtained from albino rats (8-week-old, female); the subcutaneous fat was removed carefully from the dorsal skin, leaving the dermis and epidermis, and then the skin was stored at 70 °C. The skin was thawed at room temperature and rehydrated in phosphate-buffered saline (PBS). A 2 cm  2 cm skin specimen was sandwiched between the donor and receptor of a Franz diffusion cell system, with the SC facing the donor. The receptor phase was composed of 78:20:2 (w/w/w%) PBS, ethanol, and polyoxyl (60) hydrogenated 214 castor oil (HCO-60). During the experiments, the receptor phase was stirred continuously at 150 rpm and maintained at 37 °C. The HECHA hydrogel was applied to the skin in the donor compartment. After 24 h, the skin was removed from the Franz diffusion cell, the surface of the skin was washed to remove the superfluous hydrogel, and the diffusion area was punched out. The skin tissue was incorporated with an optimum cutting temperature (OCT) embedding matrix (Cell Path Ltd., Newtown, UK) at 70 °C. The frozen skin tissue was sectioned vertically at a thickness of approximately 10 lm by using a cryotome (CM1950, LEICA, Wetzlar, Germany) at 20 °C. The excitation wavelength of the Nile red detector was 485 nm and the emission wavelength was 525 nm, respectively. All animal studies were implemented in accordance with the European Communities Council Directive No. 86/609/EEC.

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3. Results and discussion

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3.1. Gel synthesis

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The HECHA hydrogels were cross-linked using divinyl sulfone (DVS) with a molar ratio of HECHA monomer to DVS of 1:1. The final concentration of the polymer in the HECHA hydrogels was 3%. The hydroxyl groups of HEC and HA were cross-linked via divinyl groups using Michael addition at pH 13 [31]. Although DVS is a highly reactive and toxic material, the biocompatibility of hydrogels made with DVS has been confirmed in many studies [32,33]. Fig. 1 shows images of the HECHA hydrogels. FT-IR spectroscopy was used to investigate the bridging reaction between HEC, HA, and DVS. FT-IR is an effective analytical technique used to identify the functional groups, and Fig. 2 shows FT-IR spectra of the HECHA hydrogel network. The spectra were compared with those of HA and HEC; the main peaks of the HECHA hydrogels, HEC and HA were assigned in Table 1. The FTIR spectra of the HECHA hydrogels exhibited the features of the HEC and HA; however, the HECHA hydrogels also exhibited new peaks at 1287, 1325, and 1138 cm1. This indicates that DVS is involved in the synthesis of the HECHA hydrogel synthesis. In addition, the relative intensity depends on the mass ratio of HEC to HA [32–34]. These results indicate that HECHA hydrogels were formed using DVS. The morphology of the HECHA hydrogels was investigated using SEM. Cross-sectioned HECHA hydrogels exhibited macro-

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IE ð%Þ ¼

ðV 1 C 1  V 2 C 2 Þ  100; ðV 1 C 1 Þ

ð2Þ

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where V1 is the initial volume of 1,3-BG solution containing ILTG (mL), V2 is the final volume of 1,3-BG solution containing ILTG (mL), C1 is the initial concentration of ILTG (lg/mL), and C2 is the final concentration of ILTG (lg/mL). The drug release efficiency was evaluated in solutions with pH values in the range 1–13, in which the pH was adjusted using HCl and NaOH solutions. The HECHA hydrogels with incorporated ILTG were immersed in 20% 1,3-BG solution at room temperature in a darkroom for 24 h, and the concentration of ILTG in the 20% 1,3BG solution was analyzed using HPLC.

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2.7. Antimicrobial activity

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P. acnes was selected to determine the antimicrobial activity of ILTG-loaded HECHA hydrogels. The clostridial medium was prepared by changing the pH level ranging from 6 to 9. Hydrogel containing ILTG was immersed in the medium for 24 h at room temperature to release the drug. After 24 h, the hydrogel was removed, and the equivalent colony forming units (CFU) of P. acnes were incubated in the medium at 37 °C for 24 h in an anaerobic jar.

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Fig. 1. Images of HECHA hydrogels. The hydrogels are loaded according to the HEC:HA mass ratio as follows: HEC:HA (wt%) = 100:0 (HECHA10), 75:25 (HECHA31), 50:50 (HECHA11), 25:75 (HECHA13) and 0:100 (HECHA01). The white dotted line indicates the shape of the hydrogel. All hydrogels appeared transparent, and increases in the HA ratio were associated with greater transparency. HECHA: hydroxyethyl cellulose (HEC)/hyaluronic acid (HA).

Fig. 2. Fourier transform infrared (FT-IR) spectra of HECHA hydrogels in the range 500–4000 cm1 region. The spectra of the HECHA hydrogels are shown compared with those of HEC and HA standard materials. All peaks of the HECHA hydrogels exhibited similar patterns; however, new peaks related to bridging reactions were observed. From left to right: 3500–3300, 2930–2860, 1607, 1552, 1402, 1325, 1287, 1138, 1050–1030 cm1. HECHA: hydroxyethyl cellulose/hyaluronic acid.

Table 1 Assignments of infrared absorption bands from HECHA hydrogels. The synthesis was identified by characteristic absorption band of their compositions. Wave number (cm1) 3500–3300 2920 2870 1607, 1402 1552 1325, 1138 1287 1030

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Assignments (stretch) (asymmetric stretch) (symmetric stretch) (stretch) (bend) (stretch) (stretch)

AOH ACH2 ACH2 COO NAH S@O SAC CAOAC (glucose ring)

by by by by by by by by

HEC, HEC, HEC, HA HA DVS DVS HEC,

HA HA HA

HA

porous architectures with an average pore sizes in the range 50–90 lm as shown in Fig. 3. The HECHA01 hydrogel structure exhibited heterogeneous porous patterns; however, as the HEC ratio increased, the macro-porous architecture exhibited increasingly homogeneous porous patterns. These porous structures can act as water channels, so that the hydrogel can take up large quantities of water, leading to a large swelling ratio [35]. TGA provides valuable information, on the composition of multicomponent materials, thermal stability, and water content, from the change in mass of a sample as a function of temperature. In particular, the moisture content of pharmaceuticals, foods, and cosmetic products is an important feature. A TGA curve was used to assess the water binding capacity, water evaporation rate, and stability of the HECHA hydrogels. The HECHA hydrogels exhibited similar TGA curves, revealing decreases in mass from 40 °C to 140 °C, as shown in Fig. 4. This loss of mass is related to evaporation of water. At approximately 50 °C, the HECHA hydrogels lost a small

amount of water, and at about 150 °C, the mass of the HECHA hydrogels was reduced by approximately 97%. The percent mass loss at 100 °C was as follows: HECHA10 (64.59%), HECHA31 (51.48%), ECHA11 (37.97%), ECHA13 (35.27%) and (31.35%). The water did not evaporate completely at the 100 °C because of interactions between the water and the polymer chain. These results indicate that an increase in the HA ratio results in a larger water binding capacity and lower evaporation rates from the hydrogels. This is explained by considering that the HA polymer chain has more and stronger hydrogen bonding moieties than the HEC polymer chain, resulting in stronger. Moreover, the HECHA13, HECHA11, and HECHA31 hydrogels exhibited more residue related to thermal stability (approximately 5%) than HECHA10 and HECHA 01 (approximately 3%), which results indicate that the HEC and HA complex affected the stability of the HECHA hydrogels unlike HEC hydrogel (HECHA10) and HA hydrogel HECHA01 [36–38].

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3.2. Swelling ratio of HECHA hydrogels

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A general property of hydrogels is swelling when exposed to external solvents, which arises due to osmotic pressure, electrostatic forces, and a viscoelastic restoring force [6]. There have been several reports of theoretical modeling of the influence of these different forces on different scales. For example, statistical theories have been used to explain the global swelling ratio of polyelectrolyte gels, and macroscopic theories to explain the equilibrium state by minimizing the Gibbs free energy of the hydrogel [39]. Other important factors affecting the hydrogel swelling rate or swelling kinetics are the extent of the porosity, the pore size, and the structure of the porous network. Hydrogels can be classified into four groups: non-porous, with no porous network; micro-porous,

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Fig. 3. Scanning electron microscopy (SEM) images of cross-sections of the HECHA hydrogels. All hydrogels exhibited a porous structure with an average pore size in the range 50–90 lm. All images were enlarged to the same magnification (the initial magnification was 400). HECHA: hydroxyethyl cellulose/hyaluronic acid.

Fig. 4. TGA curves of the HECHA hydrogels as a function of temperature. The mass loss results related to the evaporation of water incorporated within the hydrogel. HECHA: hydroxyethyl cellulose/hyaluronic acid.

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with variable porosity and a closed-cell structure with dimensions in the range 0.01–0.1 lm; macro-porous, with variable porosity and a closed-cell structure with dimensions in the range 0.1–1 lm [40]; and super-porous hydrogels (SPHs), with dimensions of several hundred micrometers, which are connected to form an open channel system. The swelling of SPHs is due to capillary forces, which lead to rapid uptake of water that exists mainly as free water [41]. The pH-sensitive properties of the swelling behavior of HECHA hydrogels were determined by regulating the pH, as shown in Fig. 5. The swelling ratios of the HECHA hydrogels were found to vary from 1118.05% in acidic conditions to 3154.12% in alkali conditions. When comparing against pH level, the swelling ratio significantly increased by increasing the pH levels. This pHdependence is attributed due to carboxyl groups of the HA residues. The HECHA10 hydrogel was not sensitive to pH, because HEC does not have a carboxyl functional group, and had a swelling ratio of 1108.05% at pH 1 and of 1263.99% at pH 13. However, the other

Fig. 5. Swelling ratio of the HECHA hydrogels at various pH values in the range 1–13. The swelling ratio was influenced by the HEC:HA mass ratio, as well as the pH. HECHA: hydroxyethyl cellulose/hyaluronic acid.

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hydrogels (HECHA31, 11, 13, and 01) have carboxyl functional groups that show pH-sensitive properties. When increasing pH levels, the carboxyl groups of the HA residue can become ionized, forming carboxylate groups (ACOO). The negative charge of the carboxylate leads to electrostatic repulsion, increasing the pore size in the hydrogel network. Furthermore, the negatively charged group forms a hydrated membrane, which increases water binding, leading to an ability to absorb large quantities of water. In addition, HECHA hydrogels with a higher HA ratio exhibit higher swelling ratios (except at pH 1); this is because HA has superior water binding properties to HEC [8,9]. Interestingly, the increasing extent of the swelling ratio depending on pH was gradually becoming equal. This is because the functional groups were fully ionized or the ionized functional groups had a strong screening effect due to the hydrated membrane or other ions [11]. Many pH-sensitive hydrogels use polyelectrolytes and functional groups, such as carboxylic acid, sulfonic acid, or primary, secondary, and tertiary amine groups. These pH-sensitive hydrogels can be exploited to release a pharmaceutical agent at a specific pH in a controlled manner.

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3.3. Rheological and mechanical properties of HECHA hydrogels

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The rheological properties were characterized, and used to investigate the viscoelastic behavior of the hydrogels, as well as their application as transdermal delivery systems. Oscillatory tests were carried out to determine the viscoelastic properties of the hydrogels. The storage (or elastic) modulus G0 can be used to describe the elasticity or stored mechanical energy in response to stress, and the loss modulus G00 describes the viscosity or energy dissipated in response to stress; i.e.,

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0



00



G ¼ G cos d; G ¼ G sin d;

ð3Þ

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi where G ¼ G02 þ G002 , and d is the phase angle. The damping factor tan d = G00 /G0 can be used to represent the ratio of the storage and loss moduli [42]. The rheological properties of the HECHA hydrogels were investigated as function of the angular frequency, in the range 0.1– 10 rad/s, as shown in Fig. 6. The property of the HECHA10 hydrogel was not demonstrated by the rheological study because it was shattered at the experimental conditions. All the spectra revealed that the storage modulus of the HECHA hydrogels was larger than the loss modulus at all angular frequencies, and that the samples exhibited hydrogel-like behavior [43]. We find that G0 increased as the angular frequency increased, which indicates that the HECHA hydrogels formed a stable structure and did not break down during the mechanical tests [44]. Furthermore, G0 increased as the HEC:HA mass ratio increased, which indicates that the 

HEC polymer chain contributed to the strength of the HECHA hydrogels [45,46]. The mechanical properties of the HECHA hydrogels were measured using texture analysis, which is commonly used in the field of food and pharmaceutical materials. In designing a transdermal drug delivery system, the hardness, cohesiveness, and adhesiveness of the vehicle are important factors, and it is essential to maintain an appropriate balance between these factors. Table 2 lists the mechanical properties of the HECHA hydrogels. Hardness is the force required for a given deformation, i.e., the maximum peak during the first compression process. The hardness was dependent on HEC:HA ratio at a given polymer concentration. By increasing the HEC:HA ratio, the hardness of hydrogels increased by a factor of six (HECHA10 exhibited a hardness of 367.52 g, whereas HECHA01 exhibited a hardness of 59.25 g). These results are consistent with the rheological properties, which showed that the physical strength of the HECHA10 exceeded that of the HECHA01 hydrogel. Cohesiveness is the force required to maintain the shape of the sample or the ratio of the positive areas during the first and second compression. The HECHA11 hydrogel exhibited the largest cohesiveness of 1.024 g s. The HECHA10 hydrogel exhibited the largest hardness but the smallest cohesiveness; for this reason, it shattered easily and was not suitable as a drug delivery system. Adhesiveness is defined as the force required to detach the sample from a probe, or the negative force area following the first compression. Furthermore, adhesiveness plays an important role in transdermal delivery systems because skin has very dynamic movement. The HECHA13 hydrogel exhibited the highest adhesiveness of 91.58 g s. A suitable combination of HEC and HA enhances the adhesiveness of the hydrogel, which can prolong the retention time of specific skin lesions [47,48]. The HECHA13 hydrogel was chosen as the optimal material for applications as a transdermal delivery system based on these results.

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The incorporation efficiency of ILTG into HECHA13 was characterized. ILTG exhibits a lower minimum inhibitory concentration (MIC) and larger clear zone against P. acnes compared with other licorice ingredients, such as glycyrrhizin, liquiritin, liquiritigenin [27]. ILTG was dissolved in 20% 1,3-BG solution (commonly used ingredients of cosmetics) to improve the solubility of hydrophobic material [49]. The HECHA13 hydrogel exhibited an ILTG incorporation efficiency of 46.98%. The release efficiency of ILTG was determined using solutions of varying pH. There have been a number of studies described the mechanism of drug release from hydrogels for example, diffusion-controlled, swelling-controlled, and chemically controlled mechanisms [50]. However, incorporation and release of a drug occurs due to multiple factors. Diffusion-controlled release is the

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Table 2 The mechanical properties of the HECHA hydrogels, characterized using a texture analyzer. The hydrogels are labeled according to the HCE:HA mass ratio as follows: HEC:HA (wt%) = 100:0 (HECHA10), 75:25 (HECHA31), 50:50 (HECHA11), 25:75 (HECHA13) and 0:100 (HECHA01).

Fig. 6. Rheological properties of the HECHA hydrogels as determined via oscillation tests. All samples exhibited behavior. HECHA: hydroxyethyl cellulose/hyaluronic acid.

a b c

Hydrogel

Hardnessa (g)

Cohesivenessb (g s)

Adhesivenessc (g s)

HECHA10 HECHA31 HECHA11 HECHA13 HECHA01

367.52 265.18 168.60 80.95 59.25

0.96 0.97 1.02 0.97 0.93

1.56 54.66 62.34 91.57 77.60

The force required for a given deformation. The force required to maintain the sample shape. The force required to detach the sample from the probe.

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generally applicable mechanism via Fick’s first law, and depends on the pore size, degree of crosslinking, and polymer structure of the hydrogel. With swelling-controlled mechanism, the loaded drug diffuses out more rapidly from the hydrogel than via hydrogel distention. The chemically controlled mechanism is related to chemical reactions between the functional polymer groups and external factors, including pH, solvent composition, and ionic strength. Hydrogels that are pH-sensitive exhibit swelling/de-swelling behavior that depends on the pH; accordingly, pH-sensitive hydrogels can be used for therapeutic applications in which pH dependent behavior is desirable [51,52]. Fig. 7 shows the drug release efficiency as a function of pH. The ILTG release efficiency increased gradually from 41.63% at pH 1, to 55.37%, at pH 3, to 64.62% at pH 5, to 71.53% at pH 7. This increase in the release efficiency with increasing pH is similar to that of the swelling ratio. More alkali conditions result in expansion of the pore size of the hydrogel, due to electrostatic repulsion. For this reason, the drug can diffuse more easily from the hydrogel matrix. However, although the swelling ratio increased as the pH increased beyond 7 (see Fig. 5), the drug release efficiency decreased (the efficiency was 67.06% at pH 9, 57.18% at pH 11, and 40.66% at pH 13). This can be explained by considering that ILTG incorporated into the hydrogel network may destroy or deform the structure in the more basic solution. The HPLC peak intensity was used as a quantitative measure of the ILTG, which decreased above pH 9 (data not shown). Many studies have investigated the stability of phenolic compounds as a function of pH, and typically lead to deformation of the phenolic molecular structure [53,54].

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To investigate the antimicrobial activity of the HECHA13 hydrogel, containing ILTG, P. acnes was chosen because of its presence in human skin flora, as well as the induced pH imbalance. Acne lesions may be attributed to the following four processes. First, inflammatory mediators (e.g. CD4+ lymphocytes); second, alteration of the keratinization process; third, increased sebum production by androgens (sebum lipids induce interleukin 1 (IL1) secretion); and fourth, follicular colonization by P. acnes [55]. Acne is commonly treated using topical antibiotics (e.g., benzoyl peroxide or tretinoin), and acne may be combated by reducing sebum production, reversing hyperproliferation, normalizing keratinization, and clearing the microcomedones. Most studies have focused on reducing the P. acnes colonization and on reducing inflammation [56]. The pH of the SC is a crucial factor in the biophysical properties of skin that are related to acne, and the SC is normally acidic (i.e., the ‘acid mantle’ is maintained). The pH influences the growth of

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P. acnes, the production rate of exoenzymes and antigens, the stability of exoenzymes, and the enzyme activity. When culturing the P. acnes at various pH values in the range 5.5–8.5, the growth rate of P. acnes was different. P. acnes did not grow at pH 5.5, but grew well in the range 6.0–7.0 [57]. Table 3 shows the antimicrobial activity of the ILTG-loaded HECHA13 hydrogel at various pH values. To release the ILTG, the HECHA13 hydrogel was pre-incubated in a medium at various pH values. Blank groups were not pre-incubated. In the blank groups, the colony number depended on the pH. The largest number of colonies was formed at pH 7, and the number of colonies decreased as the pH increased beyond 7, although was smaller at pH 6 than at pH 9. This indicates that the pH affects the growth rate and activity of P. acnes. We speculate that the breakdown of the ‘acid mantle’ caused the growth of P. acnes, especially at pH 7. In all ILTG-loaded groups, the P. acnes colony number was smaller than in the blank groups. Superior antimicrobial activity was sound at neutral pH in all experiments. These results are consistent with those of the drug release efficiency study, which indicated that large amounts of ILTG were released at pH 7, leading to greater antimicrobial activity. For these reasons, the HECHA13 hydrogel has potential applications as a transdermal delivery system for acne lesion.

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3.6. In vitro skin permeation study using CLSM

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To investigate the potential of the HECHA hydrogels as a transdermal delivery system, confocal laser scanning microscopy (CLSM) images were used to visualize the permeation profile through the skin. The skin penetration pathways of active compounds may classified as intercellular, intracellular and follicular [58], and the follicular pathway can be used to permeate a larger amount of active compounds. Rather than ILTG, we used Nile red for the fluorescence studies. The red fluorescence intensity is an indirect indicator of the quantity of permeated ILTG. Differential interference contrast (DIC) mode was used to image the epidermis, dermis, and hair follicle layers. Table 4 shows CLSM images. The white, blue and yellow arrow indicates SC, basal layer, and hair follicles respectively. Following treatment with 1,3-BG solution containing 0.02% Nile red, fluorescence was observed at the SC in the epidermis composed of keratinocyte and melanocyte, and did not permeate to the viable layer (except SC in the epidermis). In addition, fluorescence was not observed near the hair follicle (Table 4(a)–(c)). However, the HECHA13 hydrogel group exhibited a large fluorescence intensity across the entire epidermis, and this fluorescence was distributed across in the dermis, with some decrease from the epidermis to the dermis. These results suggest that the HECHA13 hydrogel swells the SC and temporarily disturbs the skin barrier, which means that gap of corneocyte was more broadened than the 1,3BG group; therefore, the loaded hydrogel can be used to permeate the active compound into the skin through the intercellular pathway (Table 4(d)–(f)). Furthermore, the largest fluorescence intensity was observed in the hair follicle (Table 4(g)–(i)). It follows that

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Table 3 The antimicrobial activity of the HECHA13 hydrogel containing ILTG against P. acnes, which was evaluated in terms of the effect on the colony number. Colony count (CFU/mL)

Blank Experimentb

pH 6

pH 7

pH 8

pH 9

5.40  103 3.80  102

TMCa 2.80  102

1.21  104 1.58  103

1.90  104 1.72  103

a

Fig. 7. The ILTG release ratio (%) from the HECHA hydrogels at various pH values in the range 1–13. HECHA: hydroxyethyl cellulose (HEC)/hyaluronic acid (HA).

TMC: too many to count. The HECHA13 hydrogel was pre-incubated at room temperature in medium at the indicated pH. HECHA, hydroxyethyl cellulose/hyaluronic acid complex. b

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Table 4 CLSM images of cross-section of hairless the skin loaded with 1,3-BG solution labeled with a fluorescent dye, as well as the HECHA13 hydrogel applied for 24 h. The images in (d)–(i) show the same treatment group, and those in (g)–(i) show the hair follicles. The scale bar corresponds to 100 lm, the white arrow shows the SC, the blue arrow shows basal layer, and the yellow arrows show hair follicles. DIC

Nile red

Merged

1,3-BG

HECHA13 hydrogel

HECHA: hydroxyethyl cellulose/hyaluronic acid complex, 1,3-BG: 1,3-butylene glycol, DIC: differential interference contrast.

585 586 587 588 589

much of the active compound can permeate through the follicular pathway, and therefore, the HECHA13 hydrogel containing ILTG has potential application in acne control. Because the hair follicle is surrounded by sebaceous glands, which usually caused the acne, on the other hand, the ILTG effectively inhibited the acne growth.

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4. Conclusions

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We have described the synthesis of pH-sensitive hydrogels using HCE and HA for applications in clinical transdermal delivery systems for antimicrobial compounds. Breakdown of the ‘acid mantle’ is commonly involved in acne. ILTG was chosen as the drug in this study, because of its superior antimicrobial activity against P. acnes. We identified the optimal ratio of HEC to HA by investigating the physicochemical properties of the HECHA hydrogels. Hydrogel synthesis was confirmed by new peaks at 1287 cm1 (attributed to stretching vibrations of SAC), 1325 cm1, and 1138 cm1 (attributed to stretching vibrations of S@O) using FTIR. SEM images of the HECHA hydrogels revealed three-dimensional network pore sizes in the range 50–90 lm. As the HEC ratio increased, the porous structure becomes more homogeneous. The water-binding capacity of the hydrogels was evaluated using TGA. A difference in the water-binding capacity of up to 30% was

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found between the hydrogels at 100 °C (the maximum water loss was 64.59% with HECHA10, and the minimum water loss was 31.35% with HECHA01). The hydrogel swelling ratio increased with increasing pH, with the exception of HECHA10. In particular, HECHA01, which had the highest ratio of HA to HCE, exhibited the largest water uptake. This is attributed to electronic repulsion and the nature of HA. The hydrogel-like behavior of HECHA hydrogels was confirmed using rheological measurements. We found that the HECHA13 hydrogel exhibited optimal properties for transdermal drug delivery. The ILTG incorporation efficiency of HECHA13 was 46.98%, and the ILTG release efficiency was more than 70% at pH 7, due to the larger pore size, at this pH. The release efficiency decreased at pH 13, due to destruction or deformation of the ILTG structure in the basic solution. The antimicrobial activities against P. acnes were characterized as a function of pH. Colony formation of P. acnes was most active at pH 7; however, treatment with the HECHA13 hydrogel containing ILTG significantly decreased the colony number at pH 7. This result is consistent with the investigation of the release efficiency. The CLSM images reveal that the HECHA13 hydrogel easily permeated the active compound because of swelling of skin by the hydrogel, which temporally disturbed the skin barrier. In addition, the hydrogel might inhibit P. acnes growth effectively because most of the drug penetrates via the follicular pathway, which is surrounded by sebaceous glands. These results

Please cite this article in press as: S.S. Kwon et al., Physicochemical properties of pH-sensitive hydrogels based on hydroxyethyl cellulose–hyaluronic acid and for applications as transdermal delivery systems for skin lesions, Eur. J. Pharm. Biopharm. (2015), http://dx.doi.org/10.1016/j.ejpb.2015.02.025

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indicate that the HECHA hydrogels have potential applications as a transdermal drug delivery system for antimicrobial therapeutics and other skin illnesses that are caused by pH imbalance.

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Acknowledgments

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This work was carried out with the support of the ‘Cooperative Research Program for Agriculture Science & Technology Development (Project No. 008489)’, Rural Development Administration, Republic of Korea.

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