Topical and transdermal delivery with diseased human skin: passive and iontophoretic delivery of hydrocortisone into psoriatic and eczematous skin

Abstract

Psoriasis and atopic dermatitis (eczema) are both common immune-mediated inflammatory skin diseases associated with changes in skin’s stratum corneum lipid structure and barrier functionality. The present study aimed to investigate healthy, eczematous, and psoriatic excised human tissue for the effect of non-infectious skin diseases on skin characteristics (surface color, pH, transepidermal water loss, electrical resistance, and histology), as well as on permeation and retention profile of hydrocortisone. Further, differences in percutaneous absorption on application of iontophoresis on healthy and diseased skin were also investigated. Measurements of transepidermal water loss and electrical resistance showed a significant difference in psoriasis skin samples indicating a damaged barrier function. In vitro permeation studies on full-thickness human skin using vertical diffusion cells further confirmed these results as the drug amount retained in the psoriatic tissue was significantly higher when compared with the other groups. Despite no significant difference, the presence of the drug in the receptor chamber in both diseased groups can be concerning as it suggests the increased possibility of systemic absorption and adverse reactions associated with it in the use of topical corticosteroids. Application of anodal iontophoresis resulted in greater distribution of hydrocortisone into deeper layers of skin and the receptor chamber, in comparison to passive permeation. However, no significant differences were observed due to the healthy or diseased condition of skin.

Introduction

Skin diseases are among the most common illnesses affecting 30 to 70% of the population, causing a huge burden in the global context of public health [1]. The prevalence and cost associated with skin diseases exceed those of other diseases, such as diabetes and cardiovascular disease [2]. In 2010, skin disease was the fourth leading cause of nonfatal disease burden worldwide [3]. In 2013, 85 million people in the USA (i.e., 1 in 4 individuals) were reported to see a doctor for at least one skin disease, resulting in a direct health care cost of $75 billion [2]. Skin diseases can also cause psychological and social distress and have a significant negative impact on patients’ quality of life [4].

Psoriasis is a chronic T cell-mediated systemic inflammatory disease characterized by scaling dry, raised, red skin lesions (plaques) [5]. Psoriasis is a serious disease, which is associated with significant physical and psychosocial morbidity, and an overall reduction in the quality of life [6, 7]. In Europe and North America, approximately 2% of the population is affected by psoriasis, making it the most common immune-mediated inflammatory disease [8, 9]. Pharmacologic treatment strategies for management of the disease involve utilizing topical agents, such as topical corticosteroids to modulate the immune response [10].

Atopic dermatitis (AD) is a common type of eczema and is a relapsing chronic disease arising from various genetic and environmental factors [11]. It involves inflammation associated with severe itching and rash, both hallmark symptoms [12]. During the past three decades, the prevalence of AD has been continuing to increase [13]. In developed countries, it is estimated that 2–10% of adults and 15–30% of children are affected by AD [13, 14]. In the treatment of AD, topical corticosteroids are used as first-line therapy to manage the associated inflammation and pruritus [10, 15].

Topical corticosteroids are the most common prescribed drugs used in the management of both psoriasis and atopic dermatitis; however, they can cause various undesirable local side-effects including but not limited to skin atrophy, spider veins, hypertrichosis, hypopigmentation, fungal/bacterial/viral infection, and striae as well as systemic side-effects from absorption through the skin [15].

The main barrier for drug permeation through the skin is in the outermost layer of the epidermis, the stratum corneum (SC). It consists of dead corneocytes embedded in a complex multilamellar organized lipid matrix containing ceramides, cholesterol, and free fatty acids. Ceramides play a crucial role in the structuring and maintenance of the epidermal barrier function of the skin [16–18]. The lipid organization is essential to the skin’s barrier functionality as most drugs applied onto the skin permeate along these lipid regions. An altered composition and organization of ceramide profile and subsequently a reduced barrier function are often observed in skin diseases such as atopic dermatitis and psoriasis [19, 20].

There is limited information available regarding the impact of diseases on the cutaneous absorption of drugs. Due to the reasons mentioned above, it is expected that drug permeation through disrupted/compromised skin will be higher when compared with intact skin. However, the extent of increased absorption and any associated adverse risks is not known [21]. Application of passive permeation enhancement strategies such as chemical penetration enhancers on diseased skin could not only cause irritation, especially with chronic use, but could also result in an unexpected permeation profile [22–25]. This issue further extends to the patients receiving active enhancement transdermal delivery techniques such as iontophoresis for the treatment of skin disorders or other conditions. The understudied changes of rate and extent of delivery of drugs via diseased skin can result in subtherapeutic drug concentrations or potential overdosing, leading to unwanted side effects.

Iontophoresis is a long-established noninvasive technique to enhance dermal and transdermal delivery by applying a continuous low voltage current [26, 27]. Iontophoresis principally provides an electrical driving force using an electrode of the same polarity as the charge on the active molecules to transport them across SC based on electrorepulsion forces. In this process, charged (ionic) molecules are transferred via electrophoresis, while uncharged (non-ionic) and weakly charged molecules can be moved by bulk flow of the solvent (i.e., water) generated by the preferential movement of mobile cations in a phenomenon known as electroosmosis [28, 29]. Electroosmotic flow occurs in the same direction as flow of counterions from anode to cathode. Iontophoretic delivery of corticosteroids in various concentrations has been widely used for the treatment of musculoskeletal disorders, tendonitis, trismus, and psoriasis [30, 31].

The assessment of percutaneous permeation of drugs is an important step in the evaluation of dermal or transdermal delivery systems, and the most appropriate setting is the in vivo human studies, which may not be possible for ethical, practical, and economic reasons. Therefore, it is necessary to find alternative methods using appropriate surrogates for in vivo human skin [32]. Various animal models and artificial tissues have been used to study skin diseases; however, there is limited data regarding drug permeation using ex-vivo human tissue in a diseased state. Excised human skin is known to be the best surrogate model for in vivo human studies. Though it is not readily available and shows variability [32], nevertheless, in vitro investigation of healthy and disease-afflicted human skin can be of great value as the results of such study would provide a better understanding of the effect of diseases on skin characteristics as well as drug permeation and retention.

As mentioned earlier, skin diseases such as psoriasis and AD both involve an inflammatory response, which ultimately influences the skin’s lipid structure and barrier functionality. It is essential to assess how the changes in barrier function by such diseases impact skin permeability. In this study, we investigated the passive and iontophoretic delivery of topically applied hydrocortisone (a corticosteroid) using ex vivo human skin in three groups of healthy, atopic dermatitis, and psoriatic skin in order to understand the effect of skin’s disease condition on drug permeation into/across intact and diseased skin as well as a comprehensive characterization of the diseased skin.

Materials and methods

Materials

Hydrocortisone was purchased from Sigma-Aldrich (St. Louis, MO, USA). Propylene glycol was acquired from EK Industries Inc. (Joliet, IL, USA). HPLC grade methanol was obtained from Pharmco-Aaper (Shelbyville, KY, USA). Sodium phosphate dibasic salt and 10× solution of phosphate-buffered saline (PBS) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Deionized (DI) water (Milli-Q® Direct 8/16 System by MilliporeSigma, Burlington, MA, USA) was used. Silver wire (0.5-mm diameter, 99.99%) and silver/silver chloride electrodes (2 mm × 4 mm) were obtained from Fisher Scientific (Fair Lawn, NJ, USA) and A-M systems (Sequim, WA, USA), respectively. All the skin tissues were procured from the National Disease Research Interchange (NDRI) with support from NIH grant U42OD11158.

Skin preparation

Full-thickness human cadaver (healthy and diseased) skin tissues from multiple sites (abdomen, ankle and calf) from different donors, ranging 19–80 years in age, in fresh-frozen state were obtained from National Disease Research Interchange (NDRI). The full-thickness skin consisted of epidermis and the entire thickness of dermis. The subdermal fat tissue was removed using forceps and scissors, and the skin pieces were stored at − 80 °C ultra-low freezer before use. For the experimental study, skin samples were immersed into 1× PBS (an isotonic phosphate buffer saline solution containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 2 mM KH₂PO₄ with a pH of 7.4) at room temperature to thaw and then were cut into pieces using circular die punches (diameter = 22 mm, area = 3.8 cm²) for mounting onto the Franz diffusion cells. Each skin piece was weighed, and its thickness was measured using a digital material thickness gauge (MTG-DX2 by Checkline®, Cedarhurst, NY, USA).

Skin characterization

Skin morphology studies

Representative skin samples for each group were selected, and the skin surface was visualized using a handheld microscope (ProScope HR) coupled with the imaging software, ProScope HR software v 1.2.1 (Bodelin Technologies, Lake Oswego, OR, USA).

The surface of skin samples for each group was also studied using a scanning electron microscope (SEM). Skin samples were mounted on an aluminum stub using double-sided tapes, and the surface images were captured digitally and analyzed using Image-Pro (Media Cybernetics, Inc., Rockville, MD, USA).

To investigate the structure of the skin in different health conditions, histological sectioning and hematoxylin and eosin (H&E) staining was performed. Full-thickness skin pieces were immersed in Tissue-Tek® O.C.T. Compound (Sakura Finetek USA Inc., Torrance, CA, USA) and were frozen. Samples were then vertically sliced with a thickness of 20 μm using an electronic microtome cryostat (Microm HM 505 E Cryostat, Ramsey, MN, USA). Sectioned samples were then fixed and stained with hematoxylin and eosin. The prepared tissue samples were visualized on a Leica DM 750 microscope (Leica Microsystems, Wetzlar, Germany).

Skin color measurement

To determine the skin color, a handheld, portable tristimulus colorimeter (CR-400 ChromaMeter, Konica Minolta, Inc., Tokyo, Japan) was used. In this measurement, the skin surface is illuminated by a pulsed xenon lamp, and the reflected light is collected by the device, and the results are recorded using the CIE L*a*b* color space system [33]. The L*a*b* system expresses the true color of the skin as perceived by the human eye using three values: L* parameter expresses the brightness from black (0) to white (100), a* from a green (−60) to a red (+60) surface, and b* from blue (−60) to yellow (+60) [34].

Skin surface pH measurement

The pH values for the surface of skin samples were measured and recorded using a Skin pH Portable Meter (HI99181 by Hanna Instruments, Woonsocket, RI, USA) with a flat glass electrode.

Transepidermal water loss

Transepidermal water loss (TEWL) is widely used for assessing the barrier function of the skin. TEWL is defined as the amount of water vapor that diffuses across SC per skin surface per unit time. TEWL is measured using a probe with a chamber that is covered with the skin surface and contains sensors that monitor changes in relative humidity [35]. TEWL values for each skin piece were measured and recorded using a VapoMeter (Delfin Technologies Inc, Stamford, CT, USA).

Skin electrical resistance measurement

Before the permeation studies, the resistance of skin pieces was measured to ensure the integrity of the skin barrier function. For this purpose, a waveform generator, and a digital multimeter (Agilent Technologies, Santa Clara, CA, USA) connected to Ag/AgCl electrodes were used. Each skin sample was mounted on a Franz diffusion cell with both the receptor and donor chambers filled with PBS, and the skin sample was allowed to equilibrate for 15 min. Following equilibration, Ag and AgCl electrodes were inserted in the receptor and donor chambers, respectively. A load resistor (RL = 100 kΩ) was placed in series with skin, and the voltage drop across the entire circuit (V_O) and skin (V_S) was recorded on the multimeter. The skin resistance (R_S) was calculated based on the following equation [36, 37]:

$$R_S=V_S{R}_L/\left(V_O-V_S\right)$$

where V_O is 100 mV. Resistance values were calculated and reported as kΩ.

In vitro skin permeation study

In vitro permeation testing (IVPT) was performed on Franz diffusion cells. Vertical glass jacketed diffusion cells (PermeGear, Hellertown, PA, USA) with a nine mm-diameter opening (diffusion area of 0.64 cm²), flat ground joint, 5 mL receptor volume, and a stir bar (600 rpm) were used. The temperature of the receptor compartment was maintained at 37 °C using a circulating water bath connected to the water jacket around the diffusion cells providing a skin surface temperature at 32 °C. The cells were filled with 5 mL of 1× PBS as the receptor medium. Prior to this, solubility studies were performed to ensure maintaining the sink condition throughout the study. Three groups (healthy, psoriatic, and eczematous skin) were defined in this study to investigate the effect of skin health status on drug permeation and disposition (n = 6). Skin pieces were then mounted on Franz diffusion cells, with the stratum corneum facing upwards, and the donor compartment was fixed using a metal clamp. Hydrocortisone (1%w/v in propylene glycol; 50 μL) solution was applied in the donor compartment. Samples (300 μL) were withdrawn from the receptor chamber through the sampling arms at 0, 1, 2, 4, 8, 10, and 12 h and were analyzed for drug content using HPLC. Each aliquot was replaced with 300 μL of fresh 1× PBS.

At the end of the permeation study, donors were removed, and the excess formulation was wiped off the skin surface using two cotton swabs wetted with 1× PBS. Further, the diffusion area was carefully and gently wiped for 30 s in a circular motion by a cotton swab dipped in 5% lauryl ether sulfate (LES) solution. This process was repeated with a fresh cotton swab. Subsequently, the skin surface was washed with 1 mL of 1× PBS three times, while a clean donor compartment was fixed over the permeation area to facilitate the rinsing process. Finally, the skin surface was dried using two cotton swabs. After the removal of unabsorbed formulation, the skin was removed from the Franz cell and laid on a flat surface.

Iontophoresis-assisted delivery

In vitro permeation of hydrocortisone by anodal iontophoresis was evaluated using Franz diffusion cells, across full-thickness healthy and diseased (eczematous and psoriatic) excised human cadaver skin samples. In addition to the setup described in in vitro permeation study, silver (anode) and silver chloride electrodes (cathode) were placed in the donor chamber and sampling port of the receptor chamber, respectively, while ensuring the electrodes are not in contact with skin during the application of current. The electrodes were coupled in series to a source of constant current supply (Keithley 2400 Source Meter ®, Keithley Instruments Inc., Cleveland, OH, USA) and a current density of 0.5 mA/cm² was applied for 4 h, followed by passive diffusion. The total duration of the permeation study was 12 h and samples were collected at 0, 1, 2, 4, 8, and 12 h. Solubility of hydrocortisone in different ratios of deionized water and propylene glycol (1:9, 2:8, 3:7, and 4:6 v/v) was visually evaluated, and the 1:9 and 2:8 ratios were able to incorporate 1% hydrocortisone in a solution. Thus, the donor chamber was filled with 500 μL of 1% hydrocortisone solution in a modified donor vehicle for iontophoresis (0.154 M NaCl in a solution of 1-part deionized water and 4 parts propylene glycol). Passive diffusion across healthy skin type from the modified donor vehicle was also tested by keeping all parameters the same and without the application of current.

Skin extraction and drug disposition assessment

To assess the drug retention and disposition in the skin tissue, the epidermis layer (stratum corneum + viable epidermis) was carefully separated using forceps from the permeation area of the skin and was collected in a tube, and the remaining skin tissue, i.e., dermis, was also collected in a separate tube. To extract the drug, methanol was added to the tubes and the tissue was processed using a bead mill homogenizer (Bead Ruptor 24 Elite by Omni International, Kennesaw, GA, USA) using the following parameters: number of cycles: 2, speed: 6.00 m/s, time: 30 s, dwell time: 60 s. All samples were filtered using 0.22 μm PTFE filters (Millex® Syringe Filters by MilliporeSigma) and analyzed using HPLC-UV. Results were reported as mean ± SD (n = 6) for each test group.

Quantitative analyses

Skin extraction samples and in vitro permeation samples were quantified using a validated HPLC-UV method. The chromatographic analysis was performed on a Waters (Milford, MA, USA) Alliance 2695 Separations Module HPLC, equipped with a quaternary pump, and an automatic injector coupled with a photodiode array detector (Waters 996). The method was conducted using an isocratic reverse phase technique. The separation was carried out on an Agilent Eclipse Plus C18 (4.6 × 150 mm, 5 μm) column maintained at 25 °C. The mobile phase was comprised of methanol:buffer (60:40) with 20 mM Na₂HPO₄ (pH = 6 adjusted with o-phosphoric acid) used as the buffer. The flow rate was set at 1.0 mL/min, and each run-time lasted for 10 min. The volume of injection of 5 μL was used for skin extraction samples, and 20 μL for in vitro permeation samples. Matrix-matched calibration curves were used for each analysis. Peak area values were recorded at a wavelength of 245 nm. Data acquisition and processing were performed using Empower 3 software.

Data analyses

All results were reported as mean (n = 6) with standard deviation (SD) or standard error (SE). Statistical analysis was performed using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA) and Prism 8 (GraphPad Software, LLC, San Diego, CA). To compare the results of different groups, Kruskal Wallis test was performed, followed by Dunn’s multiple comparisons test. The p ≤ 0.05 was set as the criterion for statistical significance.

Results

Skin characterization

Skin morphology

The microscopic images of the skin surface, as shown in Fig. 1, show a normal brighter surface for healthy skin. Erythema and redness are observed in the AD skin type. In the psoriatic skin, thick red scaly patches (plaque) are observed.

Fig. 1.

Microscopic images of skin surface of a healthy, b eczematous, and c psoriatic skin pieces. Scanning electron microscopy (SEM) of skin surface of d healthy, e eczematous, and f psoriatic skin pieces. Microscopic cross-sectional images of g healthy, h eczematous, and i psoriatic skin tissues after H&E staining. The length of the scale bar is 200 μm in size for SEM images and 50 μm in the cross-sectional images). The presence of cell nuclei in the cornified layer (parakeratosis) is shown in (i, arrows). Tissue donor information: Healthy skin (68 years—female—abdomen), eczematous skin (52 years—female—side abdomen), psoriatic skin (52 years—female—right posterior ankle)

SEM images of the skin surface are presented in Fig. 1. A clear surface is observed for the healthy skin; however, scaling layers of SC is discernable in the eczematous and psoriatic skin.

Cross-sectional skin tissues stained by H&E technique are shown in Fig. 1. Histopathological changes in eczematous and psoriatic samples are observed. Epithelial hyperproliferation, epidermal acanthosis (thickening of viable layers), and elongated rete ridges are visible in both diseased samples but are more pronounced in psoriasis afflicted tissue. Parakeratosis (presence of cell nuclei in the cornified layer), which is also another hallmark of lesional psoriatic skin [9], is observed in psoriasis samples. Spongiosis (intercellular edema between epithelial cells) is clearly visible in eczematous skin samples.

Skin color measurement

The skin color measurements result based on CIE L*a*b* color space system are shown in Table 1. The L* parameter, which expresses the brightness of the skin surface was significantly (p ≤ 0.0001) higher in the healthy group as compared with the other groups. The a* parameter which represents the redness of the skin was significantly higher in eczematous (p ≤ 0.001) and psoriatic (p ≤ 0.05) skin when compared with healthy skin, showing more pronounced erythema in the diseased groups especially in the AD samples. No significant difference was observed in b* parameter (yellowness) across groups (Fig. 2).

Table 1.

In vitro skin color measurements based on CIE L*a*b* color space system

Skin condition	L*			a*			b*
	Mean	SD	SE	Mean	SD	SE	Mean	SD	SE
Healthy	75.58	1.21	0.50	3.05	0.38	0.16	19.19	0.84	0.34
Eczema	60.31	3.60	1.47	14.62	4.00	1.63	15.76	0.24	0.10
Psoriasis	59.33	10.19	4.16	10.91	3.56	1.45	14.99	1.29	0.53

SD Standard Deviation, SE Standard Error

Fig. 2.

In vitro skin color measurements based on CIE L*a*b* color space system (*p ≤ 0.05, ***p ≤ 0.001, ****p ≤ 0.0001)

Skin pH,TEWL, and electrical resistance assessment

The results of the initial assessment of skin samples, including pH, TEWL, and electrical resistance values, are listed in Table 2.

Table 2.

Skin pH, TEWL, and electrical resistance measurement results

Skin condition	pH (n = 6)			TEWL (g/m2 h) (n = 6)			Resistance (kΩ) (n = 6)
	Mean	SD	SE	Mean	SD	SE	Mean	SD	SE
Healthy	5.67	0.63	0.26	5.10	0.40	0.16	198.22	53.60	21.88
Eczema	5.62	0.49	0.20	8.40	1.97	0.80	149.40	38.93	15.89
Psoriasis	5.04	0.49	0.20	15.00	4.92	2.01	4.38	2.66	1.08

TEWL transepidermal water loss, SD standard deviation, SE standard error

The skin surface pH measurements showed no significant difference between groups, as shown in Fig. 3. However, a significant increase in TEWL measurements was observed in the psoriasis group when compared with the healthy (p ≤ 0.001) and eczema (p ≤ 0.01). There was also a significant decrease in skin resistance in the psoriasis group when compared with healthy (p ≤ 0.0001) and eczema (p ≤ 0.001) groups confirming a compromised barrier function in psoriatic skin.

Fig. 3.

a Skin surface pH, b transepidermal water loss (TEWL), and c electrical skin resistance measurements of healthy, eczematous, and psoriatic tissues (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001)

In vitro passive permeation

The permeation profile of hydrocortisone through full-thickness human skin is shown in Fig. 4. At the end of 12 h, no drug was found in the receptor compartment of the healthy skin group. While there were no significant differences in comparison to healthy skin tissue, cumulative amount (mean ± SE) of 0.92 ± 0.03 μg/cm² for eczematous and 3.48 ± 2.64 μg/cm² for psoriatic samples was observed in the receptor compartment after 12 h. The steady-state flux of hydrocortisone across eczematous and psoriatic skin was found to be 0.34 ± 0.00 μg/cm²/h and 0.79 ± 0.61 μg/cm²/h, respectively.

Fig. 4.

a Permeation profile and b transdermal flux of hydrocortisone (1% solution in propylene glycol) obtained on in vitro evaluation across full-thickness human skin (healthy, eczematous and psoriatic)

The results of hydrocortisone extracted from skin samples are shown in Fig. 5. The results of the drug amount were normalized based on both skin weight and area. In both calculations the amount of drug found in psoriatic tissue was significantly (p ≤ 0.01) higher than healthy skin. The amount of drug in healthy epidermis was significantly (p ≤ 0.0001) higher than healthy dermis, while no significant difference was observed in other groups.

Fig. 5.

Topical delivery of hydrocortisone (1% solution in propylene glycol) obtained on in vitro evaluation across full-thickness human skin (healthy, eczematous and psoriatic). Amount of hydrocortisone extracted from the skin tissue normalized by a skin weight and b skin area. c Disposition of hydrocortisone in different layers of skin (**p ≤ 0.01, ****p ≤ 0.0001)

Effect of drug vehicle change in iontophoresis-assisted delivery

A current density of 0.5 mA/cm² was sustained by the modified donor vehicle (0.154 M NaCl in a solution of 1-part deionized water and 4 parts propylene glycol) for the duration of application (4 h). Similar to passive diffusion from propylene glycol, delivery from the modified vehicle resulted in no systemic delivery (0.00 ± 0.00 μg/cm² in the receptor compartment). There was no significant difference (p > 0.05) in the amount of hydrocortisone delivered to skin from the modified vehicle (2.29 ± 0.22 μg/cm²), in comparison to delivery from propylene glycol (1.44 ± 0.22 μg/cm²). Data normalized by weight showed no significant difference either (p > 0.05). Further, greater distribution into the epidermal layers of skin (100.00 ± 0.00%) in comparison to the dermis was observed (Fig. 6).

Fig. 6.

a Permeation profile of hydrocortisone comparing 1% solution in propylene glycol (PG) and modified vehicle for iontophoresis (Aq-PG: 0.154 M NaCl in 1:4 deionized water and PG) obtained on in vitro evaluation across full-thickness healthy human skin. b Amount of hydrocortisone extracted from the skin tissue normalized by skin weight and skin area. c Disposition of hydrocortisone in different layers of skin (*p ≤ 0.05, **p ≤ 0.01, ”ns” p > 0.05)

Effect of iontophoresis on percutaneous absorption of hydrocortisone

Anodal iontophoresis (0.5 mA/cm²) was successfully applied for 4 h, and hydrocortisone (7.91 ± 0.26 μg/cm²) was absorbed into skin and the receptor compartment across healthy human skin. However, there was no significant difference (p > 0.05) in comparison to passive permeation. Although the total amount of drug absorbed (into skin and in receptor compartment) was found to be significantly higher (p ≥ 0.05) with iontophoresis on eczematous (40.22 ± 8.98 μg/cm²) and psoriatic skin (34.99 ± 5.66 μg/cm²) in comparison to passive diffusion across healthy skin (1.44 ± 0.30 μg/cm²), there was no significant difference (p > 0.05) in comparison to passive diffusion across eczematous (3.49 ± 0.14 μg/cm²) and psoriatic skin (35.29 ± 17.35 μg/cm²) respectively (Fig. 7).

Fig. 7.

Percutaneous absorption (average cumulative amount absorbed into skin and in receptor compartment) of hydrocortisone (1%) obtained by in vitro evaluation across full-thickness human skin (healthy, eczematous, and psoriatic). Passive diffusion from propylene glycol (PG) or modified donor vehicle (Aq-PG: 0.154 M NaCl in 1:4 deionized water and PG) compared with anodal iontophoresis (ITP; 0.5 mA/cm²) (*p ≤ 0.05, “ns” p > 0.05)

There was no significant difference (p > 0.05) in the amount of hydrocortisone delivered to the receptor compartment with iontophoresis for healthy (2.05 ± 0.11 μg/cm²), as well as diseased skin (7.11 ± 1.10 μg/cm² across eczematous skin; 16.93 ± 1.41 μg/cm² across psoriatic skin) in comparison to passive diffusion across respective skin groups. Iontophoresis across diseased skin, nevertheless, enhanced delivery to receptor compartment in comparison to passive diffusion across healthy human skin (p ≤ 0.05; Fig. 8).

Fig. 8.

a Permeation profile of hydrocortisone from 1% solution in modified vehicle for iontophoresis (Aq-PG: 0.154 M NaCl in 1:4 deionized water and PG) obtained on in vitro evaluation across full-thickness human skin. Iontophoresis (ITP) applied for 4 h, followed by passive diffusion across healthy, eczematous, and psoriatic skin. b Average cumulative amount delivered in receptor compartment after 12 h compared with passive diffusion from propylene glycol (PG) as vehicle (*p ≤ 0.05, **p ≤ 0.01, “ns” p > 0.05)

Topical delivery of hydrocortisone was not significantly increased with iontophoresis (p > 0.05, Fig. 9). However, for healthy skin groups, distribution of drug into the layers of skin was affected by iontophoresis where the percent drug absorbed in epidermis was significantly higher (p ≤ 0.05) than dermis for passive diffusion, but statistically similar (p > 0.05) with iontophoresis (57.71 ± 2.35% in epidermis; 42.29 ± 2.35% in dermis, Fig. 10).

Fig. 9.

Topical delivery of hydrocortisone (1%) obtained on in vitro evaluation across full-thickness human skin (healthy, eczematous, and psoriatic) on passive diffusion from propylene glycol (PG) or modified donor vehicle (Aq-PG: 0.154 M NaCl in 1:4 deionized water and PG) in comparison to anodal iontophoresis (ITP; 0.5 mA/cm²). Amount of hydrocortisone extracted from the skin tissue normalized by a skin area and b skin weight. c Disposition of hydrocortisone in different layers of skin (*p ≤ 0.05, **p ≤ 0.01, ”ns” p > 0.05)

Fig. 10.

Percentage distribution to a epidermis and dermis of skin and b receptor compartment receptor compartment and skin on percutaneous absorption of hydrocortisone (1%) obtained by in vitro evaluation across full-thickness human skin (healthy, eczematous, and psoriatic). Passive diffusion from propylene glycol (PG) or modified donor vehicle (Aq-PG: 0.154 M NaCl in 1:4 deionized water and PG) compared with anodal iontophoresis (ITP; 0.5 mA/cm²) (***p ≤ 0.001, “ns” p > 0.05)

Quantitative Analysis

A simple and rapid RP-HPLC method was successfully developed and validated for hydrocortisone. Chromatographic conditions were optimized to achieve a sharp, symmetrical peak with reasonable retention time. The total run time was short (10 min), and HC’s peak was achieved at the retention time of 5.1 min. Specificity was confirmed as no interfering peak from the matrix was observed at or near the analyte’s retention time. An eight-point calibration curve was constructed using calibration standards prepared in matrix-matched solutions. Linearity (R² ≥ 0.9999) was established for all calibration curves in the concentration range of 0.05–100 μg/mL. The slopes and intercepts were calculated from the plot of peak area versus concentration. The limit of detection (LOD) and limit of quantification (LOQ) were calculated by the following equations:

$$\text{LOD}=3.3\sigma /S$$

$$\text{LOQ}=10.0\sigma /S$$

where σ is the standard deviation of intercept values and S is the slope acquired from drawn calibration curves.

The calculated LOD and LOQ values were 0.07 and 0.21 μg/mL, respectively, confirming that the method had sufficient sensitivity. Accuracy and precision were determined by six injections of three levels of concentration for intra-day and total injection of nine on three separate days for the inter-day validation. The intra- and inter-day precision (%CV) at all concentration levels were found to be less than 5%. Accuracy values for all the drugs were found to be within the range of 99.55–109.78% (Table 3). All the validation parameters were shown to be within the specified limits. The developed HPLC method was specific, sensitive, accurate, precise, and reproducible, hence was suitably employed to assay the hydrocortisone amount in in vitro permeation and skin extraction samples.

Table 3.

Validation parameters (inter- and intra-day precision and accuracy) of the HPLC method

Expected conc. (μg/mL)	Intra-day (n = 6)			Inter-day (n = 9)
	Measured conc (μg/mL, mean ± SD)	Accuracy (%, mean ± SD)	%CV	Measured conc (μg/mL, Mean ± SD)	Accuracy (%, mean ± SD)	%CV
0.1	0.11 ± 0.01	111.17 ± 5.13	4.62	0.11 ± 0.01	109.78 ± 5.05	4.60
1.0	1.01 ± 0.00	101.14 ± 0.18	0.17	1.01 ± 0.00	102.61 ± 0.29	0.29
25	24.89 ± 0.02	99.55 ± 0.07	0.07	24.91 ± 0.05	101.34 ± 0.19	0.19

Conc. concentration, SD standard deviation, CV coefficient of variation

Discussion

Atopic dermatitis (AD) and psoriasis are the most common inflammatory skin diseases with a significant impact on the skin’s structure and barrier functionality [38]. Skin compromised by mechanical damage and removal of SC through tape stripping has been used as a model for a diseased state of the skin [39, 40]. Such techniques can increase TEWL values, as seen similarly in various dermatoses. However, they cannot fully represent the disease condition in which a combination of endogenous and exogenous factors is involved in the inflammatory process. Artificial and reconstructed skin models to mimic disease conditions have also been investigated, though they are not capable of representing the multitude of in vivo skin properties [41]. To this date, there is limited data regarding drug permeation using ex-vivo human tissue in a diseased state. Regardless of its shortage and variability, excised human skin is considered to be the closest surrogate model for in vivo human studies [32]. Hence, in this study, we investigated the in vitro skin characteristics as well as permeation and retention profile of hydrocortisone in healthy and disease-afflicted human skin to better understand the effect of such diseases on skin’s structure and behavior.

Erythema (redness) of the skin surface is one of the most apparent clinical signs of AD due to the disease’s inflammatory nature. In our study, the skin’s erythema was clearly observed in microscopy imaging of eczematous skin samples. Psoriatic skin samples exhibited thick red patches covered with silvery scales, which is a classic symptom of the disease. SEM imaging of the skin’s surface showed the raised loose corneocytes in diseased samples when compared with the healthy tissue showing a perturbed SC and desquamation henceforth defect in the skin barrier. Epidermal hyperplasia (thickening of viable layers) is a histological hallmark observed in both AD and psoriasis [42]. The results of our histological studies also showed similar behavior in afflicted skin; however, the extent of thickening was not quantified due to limited availability of diseased skin tissue samples from multiple donors. Epidermal hyperplasia is known to be a result of a barrier disruption and takes place as part of a series of homeostatic processes aimed at barrier restoration [43, 44]. Elongated epidermal rete pegs (epithelial extensions which project into dermis) and parakeratosis (presence of nuclei in SC) were markedly visualized in psoriatic skin samples.

Regarding skin color, as measured by a ChromaMeter, significantly increased L* values were observed for healthy skin samples when compared with the diseased skin samples suggesting a brighter skin surface. The a* values were found to be significantly higher in eczematous and psoriatic samples due to the rash and redness (erythema) in the skin. However, no significant difference was observed in b* values across groups. The L* and a* values both seem to be afflicted by the diseased conditions associated with the inflammatory response, which has an impact on the skin’s structure and, subsequently, drug permeation into/across the skin. Hence, it seems that a visual as well as chromametric assessment of skin can provide an appropriate predictor of potential structural skin changes caused by inflammation. While TEWL and skin conductance measurements are extensively used tests for initial assessment of skin’s integrity prior to an IVPT study [45], a chromametric evaluation appears to be an additional critical tool to assess the integrity of deeper layers of the skin especially in cases that the skin disease is clinically undiagnosed.

Skin’s surface is known to have an acidic nature with a pH ranging from 5.4 to 5.9 [46]. The pH of the skin surface can be influenced by numerous physiological and external factors, as well as diseases [47]. While it is reported that skin surface pH is higher in patients with atopic dermatitis than in healthy controls [48], however, in our in vitro study, we did not observe any significant pH changes in the eczematous skin samples. In case of psoriasis, there is conflicting information regarding pH changes on the skin surface. Cannavo et al. reported that the skin surface pH is significantly lower in psoriatic patients than in healthy subjects attributing it to the metabolic modification of keratinocytes and elevated production of fatty acid mediators involved in immune response and inflammation [49]. In another study performed by Delfino et al., no difference in pH values was identified between psoriasis patients and the control group [50]. Similarly, we did not find any significant difference between psoriatic and healthy skin surface pH values.

The in vitro measurement of TEWL has been used as an accepted quantitative parameter to assess skin barrier integrity and function [35]. It has been suggested that there is a direct correlation between TEWL values and skin permeability of compounds [51]. The intact SC of healthy skin impedes the free passage of water by its barrier function, resulting in a low TEWL value [52]. However, there is previous evidence that it is raised in lesional and non-lesional skin in subjects with AD [53, 54] as well as psoriasis [55, 56]. Our results similarly showed a significant increase in the TEWL values measured in psoriatic skin samples indicating a weakened barrier function. In our study, we were able to see the similar behavior of in vitro excised human psoriatic skin samples as compared with the previous in vivo studies.

Skin electrical resistance measurement is another quick and reliable method for skin integrity test [57]. In this test, the electrical resistance of the skin is measured by the application of a small electrical potential across the skin sample. In case of a disrupted barrier, the lower electrical resistance is expected to be observed. Our results showed that psoriatic skin had significantly lower electrical resistance, which is in accordance with the TEWL data confirming a damaged SC. The AD samples did not show any significant differences in TEWL and electrical resistance measurements, which shows there was no significant barrier dysfunctionality present in the samples studied. The results shown here are limited to samples studied and cannot be generalized due to patient intrinsic/extrinsic factors and disease severity amongst population. Hence, based on these results, it cannot be concluded that eczematous skin behaves in the same manner as healthy tissue since the inflammation can have a significant impact on the structure of deeper layers of the skin and, consequently, on permeant’s disposition and retention in skin.

Previous in vivo human studies suggest a modest increase in penetration through damaged or diseased skin compared with intact skin attributed to the changes in the lipid structure and organization in SC. Limited data and inconsistent results may be explained by the lack of availability of diseased skin tissue and variability in skin damage [58, 59]. The previous data regarding penetration through eczematous skin mostly shows that AD leads to increased chemical absorption. Similarly, it has been shown that skin penetration is often increased in psoriatic skin [59].

Hydrocortisone was selected as the permeant in this study on account of its passive permeability owing it to its low molecular weight and moderate lipophilicity (logP = 1.6) and also as a representative of its family that is corticosteroids, which are widely used for management of AD and psoriasis. In our IVPT results, drug was found in the receptor chamber following passive diffusion for both disease groups, while there was no drug present in the healthy group. The drug amount in the receptor is often considered a measure of dermal absorption, which sets the drug pharmacokinetics in the systemic compartment circulation. Although no significant difference was observed between the diseased and the healthy group, there is a higher level of absorption in diseased skin following topical administration of medications which can result in an unintended clinical outcome. As most AD and psoriasis treatments are based on topical therapy, excessive systemic absorption can be of concern, especially in case of corticosteroids, with a long-known list of adverse reactions [60]. The calculated passive steady-state flux values in psoriatic samples were 2.3-fold higher than the AD group.

Following IVPT, skin extraction was performed to assess the drug amount in epidermis and dermis to further understand the effect of disease state on drug disposition behavior. Our results showed that the drug amount in the healthy epidermis following passive diffusion was significantly higher than healthy dermis, while no significant difference was observed in the diseased skin groups. This finding indicates that in diseased skin, the drug further disposes to the deeper layers of skin (dermis) while it tends to be more accumulated in the superficial layers of healthy tissue. Furthermore, the comparison of total drug amount also showed that the drug amount retained in psoriatic skin was significantly higher than the other groups. This finding is in complete accordance with the TEWL and electrical resistance data, proving once more the damage in skin barrier in diseased state can significantly affect the drug permeation and disposition in skin tissue.

Despite several reports of enhanced dermal absorption of drugs and benefits of topical treatment of local cutaneous effects, there have been limited applications of iontophoresis in the treatment of dermal conditions [27, 59]. The ability of iontophoresis to enhance dermal absorption is often offset by complications associated with incorporating an external energy source that is required to electronically control the drug delivery system. The first-generation iontophoretic devices were found to be too bulky and expensive for portable use. However, over the years, advancements in microelectronics have paved the way for newer, patient-compliant designs [60, 61]. Studies performed on diseased skin models suggest percutaneous absorption through clinically diseased skin can be enhanced only to a certain extent in comparison to intact skin, if at all. However, severity, site of testing, and type of clinical disease may affect observations greatly, resulting in conflicting reports [53]. Additionally, different parameters used for physical and chemical enhancement may further affect drug delivery and disposition [27, 59].

Although hydrocortisone (pK_a 12.59) remains uncharged at a pH range of 0 to 10 [63], and is not subject to electromigration, iontophoretic delivery was studied for this molecule as enhancement can still be achieved by electroosmosis. Enhanced delivery of hydrocortisone across healthy skin samples have been reported by iontophoresis and the use of surfactants and cyclodextrins for greater solubility of hydrocortisone. The effect of pH and hydrolysis of different salt forms of the drug on application of electrical current have also been studied [27, 64–67]. However, the effect of iontophoresis on the percutaneous delivery of hydrocortisone has not been investigated for in vitro diseased human skin samples to the best of our knowledge.

The current density of 0.5 mA/cm² has been reported to be well tolerated for a duration of 4 h in addition to demonstrating enhanced percutaneous delivery of several chemicals, and was hence, selected as the treatment parameter in our study [27, 68–70]. The donor solution for IVPT was modified for iontophoretic delivery to ensure sustenance of selected current density for the duration of application. The solubility of 1% hydrocortisone was tested in different ratios of deionized water and propylene glycol solutions, and the highest aqueous ratio that could incorporate hydrocortisone without precipitation was found to be 20% v/v. Thus, 0.154 M NaCl in 1:4 deionized water and propylene glycol was selected as the modified vehicle. Passive diffusion from the modified vehicle was compared with delivery from propylene glycol, and similar topical and transdermal delivery was observed. Additionally, the drug distribution between the epidermis and dermis was found to be similar as well (Fig. 6). Thus, changes in percutaneous delivery in the iontophoresis-mediated groups using the modified donor solution were solely attributed to the application of current.

Enhanced delivery with anodal iontophoresis was observed with healthy as well as diseased skin. The total amount of hydrocortisone absorbed into skin and the receptor compartment was enhanced approximately 3 folds for healthy skin and 12 folds for eczematous skin, in comparison with passive diffusion in respective skin types. In the healthy skin samples, the application of iontophoresis acted as a driving force to deliver hydrocortisone into the deeper layers of skin and drug was observed in the receptor compartment after 12 h. Although the total amount of hydrocortisone delivered into skin was statistically similar to passive diffusion, the percentage delivery into dermis and the receptor compartment was increased. In the psoriatic skin samples, passive diffusion of hydrocortisone resulted in higher drug delivery than eczematous and healthy skin samples. It also resulted in greater distribution of hydrocortisone into the dermis and receptor compartment, in comparison to passive diffusion. Similarly, for eczematous skin, delivery into skin was found to be higher with deeper distribution as more drug was found in the dermis with iontophoresis, in comparison with the drug distribution obtained with passive diffusion.

Previous studies have established that a high concentration of hydrocortisone can be maintained in the stratum corneum; however, it does not guarantee therapeutically relevant drug amounts in the epidermis and dermis of skin [71, 72]. Thus, greater bioavailability with iontophoresis, as observed in the deeper layers of skin our study, shall be more beneficial than the slow release of hydrocortisone at sub-therapeutic levels from a reservoir formed in the stratum corneum of skin [71–73]. However, the current density applied and the duration of application can greatly affect the distribution of hydrocortisone into and across skin [27, 74]. Systemic administration of corticosteroids is recommended only in the case of acute dermatological disorders or chronic diseases as they can exhibit many side effects [75]. Iontophoresis-mediated delivery of hydrocortisone can help modulate delivery based on the disease condition [27, 74]. Although parameters of iontophoresis selected in our study is regarded as safe for application on healthy human skin, preexisting skin lesions or uneven contact due to the disease condition could offer low-resistance pathways and form a point of burn initiation. This can be avoided by careful positioning of the iontophoresis electrodes and by maintaining a uniform, tight contact with skin [76]. Lastly, solubility of hydrocortisone in the vehicle applied on skin can also affect its percutaneous absorption and distribution [62, 63].

Conclusion

In this study, we investigated three types of excised human skin tissues, including healthy, atopic dermatitis, and psoriasis. The effect of these disease conditions on skin characteristics, in vitro passive and iontophoretic delivery, and skin retention upon topical administration of hydrocortisone was evaluated. Both psoriatic and eczematous skin showed significant erythema compared with healthy skin. Reduced barrier functionality was observed in psoriatic skin with significantly higher TEWL values and decreased electrical resistance measurements. Drug amount retained in psoriatic skin, was significantly higher compared with normal skin, suggesting that barrier dysfunction and the structural changes in afflicted skin can have a substantial effect on drug permeation profile into/across diseased skin. As the physiochemical properties of many drug molecules limit their passive permeation, methods to enhance transdermal transport such as iontophoresis discussed here can help augment the transdermal flux through healthy as well as diseased skin. Subsequent uptake by dermal microcirculation and eventual uptake into the systemic circulation can facilitate drug molecules to elicit their pharmacodynamic action [74]. Additionally, drugs that are required to elicit their action only within the skin compartments, including hydrocortisone, may also benefit from such enhancement techniques as the treatment of certain skin diseases can be achieved by targeting drug delivery into the local tissue [22, 23, 68]. In case of iontophoresis, by carefully controlling its parameters, wider distribution into skin can be achieved to elicit the required pharmacodynamic response (e.g., reduced inflammation with enhanced delivery of hydrocortisone into the layers of skin) [77, 78]. However, the effect of iontophoresis and other enhancement techniques would differ based on the physiochemical properties of the drug being delivered as its permeation through skin can occur via different routes and would need to be evaluated on an individual basis.

Data Availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.