Enhanced transdermal delivery by combined application of dissolving

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Enhanced transdermal delivery by combined application of dissolving microneedle patch on serum-treated skin Suyong Kim, Manita Dangol, Geonwoo Kang, Shayan F. Lahiji, Huisuk Yang, Mingyu Jang, Yonghao Ma, Chengguo Li, Sang Gon Lee, Chang Hyun Kim, Young Wook Choi, So Jeong Kim, Ja Hyun Ryu, Ji Hwoon Baek, Jaesuk Koh, and Hyungil Jung Mol. Pharmaceutics, Just Accepted Manuscript • Publication Date (Web): 27 Apr 2017 Downloaded from http://pubs.acs.org on April 28, 2017

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Molecular Pharmaceutics

Enhanced transdermal delivery by combined application of dissolving microneedle patch on serum-treated skin

Suyong Kim†,‡, Manita Dangol†, Geonwoo Kang†, Shayan F. Lahiji†, Huisuk Yang†, Mingyu Jang†,‡, Yonghao Ma†, Chengguo Li†, Sang Gon Lee§, Chang Hyun Kim§, Young Wook Choi§, So Jeong Kimǁ, Ja Hyun Ryuǁ, Ji Hwoon Baekǁ, Jaesuk Kohǁ, and Hyungil Jung*,†,‡ †

Department of Biotechnology, Building 123, Yonsei University, 50 Yonsei-ro, Seodaemun-

gu, Seoul 03722, Korea ‡

Juvic Inc., Building 102, Yonsei Engineering Research Park, 50 Yonsei-ro, Seodaemun-gu,

Seoul 03722, Korea §

College of Pharmacy, Chung-Ang University, 84 Heuksuk-ro, Dongjak-gu, Seoul 06974,

Korea ǁ

Dermapro Skin Research Center, Dermapro Ltd., 30 Bangbaejoongang-ro, Seocho-gu, Seoul

06684, Korea *

Corresponding author: Hyungil Jung. Address: Department of Biotechnology, Yonsei

University, 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. Tel: +82-2-2123-2884. Fax: +82-2-362-7265. E-mail: [email protected]

ACS Paragon Plus Environment

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

ABSTRACT Dissolving microneedle (DMN), a transdermal drug delivery system in which drugs are encapsulated in a biodegradable polymeric micro-structure, is designed to dissolve after skin penetration and release the encapsulated drugs into the body. However, due to limited loading capacity of drugs within micro-sized structures, only small dosage can be delivered which are often insufficient for patients. We propose a novel DMN application that combines topical and DMN application simultaneously to improve skin permeation efficiency. Drugs in pre-treated topical formulation and encapsulated drugs in DMN patch are delivered into the skin through micro-channels created by DMN application; thus, greatly increasing the delivered dose. We used 4-n-butylresorcinol to treat human hyper-pigmentation, and found that sequential application of serum formulation and DMNs were successful. In skin distribution experiments using Alexa Fluor 488 and 568 dyes as model drugs, we confirmed that the pre-treated serum formulation was delivered into the skin through micro-channels created by the DMNs. In vitro skin permeation and retention experiments confirmed that this novel combined application delivered more 4-n-butylresorcinol into the skin than traditional DMN-only and serum-only applications. Moreover, this combined application showed a higher efficacy in reducing patients’ melanin index and hyper-pigmented regions, compared with the serum-only application. As combined application of DMNs on serum-treated skin can overcome both dose limitations and safety concerns, this novel approach can advance developments in transdermal drug delivery.


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TABLE OF CONTENTS GRAPHIC

KEYWORDS Dissolving microneedle, Topical formulations, Drug delivery, Micro-channels, Serum application, Solid microneedle, Hyper-pigmentation



INTRODUCTION Skin-mediated topical drug delivery, such as application of ointments, gels, creams, or cosmetic serums, has been used to treat various skin diseases.1-4 In this topical delivery route, liquid formulations containing drugs are applied to the skin, and are intended to diffuse into the body. However, the most active compounds in the formulations cannot penetrate the stratum corneum.5,6 Although topical application is patient-friendly and safe, low rates of skin penetration can limit the efficacy of the active compounds. To overcome this issue, microneedles that can pierce the skin have been conceptualized and developed.7,8 Previously, solid microneedles (SMN) made of metal or silicon were developed to create micro-channels in the skin.9-11 These channels allowed application of topical formulations before or after drug treatment to improve the efficacy of drug absorption into the body.12-14 However, SMNs are typically made of permanent materials, leading to safety concerns. Potentially serious side effects of SMN use include infection caused by exposure to air or sunlight immediately after treatment, which results in irritation, redness, and red spots on the application site.15 Moreover, SMNs are hazardous wastes, raise concerns of patient safety and inappropriate or accidental reuse as traditional hypodermic needles.16 In contrast to SMNs, dissolving microneedles (DMN), where drugs are encapsulated in a biodegradable polymeric micro-structure, are designed to dissolve after skin penetration and release the drugs into the body during dissolution.17-19 Although this novel transdermal drug delivery system increases the permeability of drugs through the skin barrier, only small quantities of drugs can be delivered due to limited loading space in the micro-dimension of DMNs;20 therefore, efficacy to patients is limited. To overcome this limitation, hydrogelforming microneedle technology comprising swellable microneedles for skin channel creation and a reservoir for drugs has been developed.21-23 In this system, however, the


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backbone material of the swellable microneedle is not biodegradable.24 Moreover, the drug formulated in the extra reservoir requires long application time, rendering the formulation inconvenient to patients, as the delivery time of the drug is based on the diffusion kinetics. Therefore, a novel simplified microneedle approach that combines increased delivery efficacy and patient safety is required. Since solidified DMNs with a high mechanical strength can create skin channels by disrupting the stratum corneum, DMNs could replace SMNs without compromising patient safety. In addition, simultaneous topical application, which acts as drug reservoir, can deliver sufficient quantities of drugs via DMN channels. In this way, DMN application after topical formulation treatment could overcome DMN dose limitations, because the drugs in both encapsulated DMNs and topical formulations are absorbed through skin channels simultaneously. Although a combination of these applications would improve the quality and efficiency of drug delivery, the combined application of DMN and topical formulations has not been described in the literature. In this study, we applied a DMN patch after topical serum treatment by using 4-nbutylresorcinol, the safety and efficacy of which have been approved in treating human hyper-pigmentation.25 To find the optimal conditions for our combined application method, the appropriate time interval between serum treatment and DMN patch application was investigated. The efficacy of transdermal delivery in this novel combined application was visualized using Alexa Fluor 488 and 568 dyes as model drugs. Further, in vitro skin permeation and retention of 4-n-butylresorcinol were compared with those in control serumonly and DMN-only groups. Moreover, we confirmed the efficacy of 4-n-butylresorcinol in the combined group in a randomized clinical study by assessing the melanin index and hyperpigmented region. This combined application including DMN application after topical



treatment offers a novel microneedle approach for enhanced transdermal delivery without apparent safety issues.


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EXPERIMENTAL SECTION Materials. Hyaluronic acid (HA; 28.5 kDa and 490 kDa, Primalhyal 50) was purchased from Soliance (Pomacle, France). 4-n-butylresorcinol was purchased from Seojin Biotech (Yongin, Korea). 1,3-Butylene glycol was purchased from KH Neochem Co., Ltd. (Tokyo, Japan). Carbopol 934P was kindly gifted by Masung Co., Ltd. (Seoul, Korea). Tinocare

GL (scleroglucan

polysaccharide)

was

generously

provided

by

BASF

(Ludwigshafen, Germany). Tego Care 450 (polyglyceryl-3-methyl glucose distearate), mineral oil, grape seed oil, acetyl alcohol, and stearic acid were kindly provided by Interlees Ltd. (Seongnam, Korea). Olivem 1000 (sorbitan ester and cetearyl ester of olive oil fatty acids) was provided by B&T (Milan, Italy). Urea was purchased from Daejung Chemicals (Si-heung, Korea). Carnosine was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). Alexa Fluor 488 and 568 (hydrazide, sodium salt) dyes were purchased from Life Technologies (Carlsbad, CA, USA). Hair-removed pig cadaver skin (1-mm thickness) was purchased from Cronex (Hwaseong, Korea). Trypan blue solution (0.4%) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hairless mouse dorsal skin (SKH:HR-1, five-week-old) was purchased from Orient Bio (Seongnam, Korea). All chemicals and reagents obtained from commercial sources were analytical-grade. Triple-distilled water was used for all experiments. Fabrication of 4-n-butylresorcinol DMN patch. DMN patches for 4-nbutylresorcinol were fabricated as described previously.25 Briefly, 5.55% w/w 4-nbutylresorcinol, 3.37% w/w 1,3-butylene glycol, 12.47% w/w HA (28.5 kDa), and 1.9% w/w HA (490 kDa) were dissolved in distilled water. The mixture was homogenized using a planetary centrifugal mixer (ARV-310, Thinky Corporation, Tokyo, Japan), and droplets were placed on a hydrocolloid patch using a dispenser (Musashi Co., Tokyo, Japan). The droplets



were then elongated into biconcave shape and formed DMN arrays. In total, 140 microneedles were fabricated, and their 4-n-butylresorcinol content was approximately 120 µg per patch. Alexa Fluor 488 was mixed with the same portion as 4-n-butylresorcinol in distilled water, and the same process was used to encapsulate it in the DMNs. Fabrication of 4-n-butylresorcinol serum. An aqueous phase consisting of hydrogel base and humectants was homogenized with a melted oil phase consisting of emulsifier, emollient, and lipids by using a homogenizer (Ultra-Turrax T25; IKA, Staufen, Germany) at 13,000 rpm for 10 min. For the hydrogel base of the aqueous phase, 0.5% w/v Carbopol 934P was dissolved in distilled water with 5% v/v glycerin and 0.5% w/v carnosine. Next, 0.3% w/v 4-n-butylresorcinol and humectants containing 10% w/v Tinocare GL and 3% w/v urea were added prior to homogenization. For the oil phase, 0.5% w/v Olivem 1000 as a nonirritant emulsifier and 4% v/v grape seed oil as an emollient were mixed, then 2% v/v stearic acid and 1% v/v acetyl alcohol were added to confer rheological properties and increase occlusiveness. The dispersions were neutralized with carnosine in the aqueous phase. The homogenized serum was degassed and cooled to 25°C. Alexa Fluor 568 as a model drug was used with the same portion of the serum instead of 4-n-butylresorcinol. Serum with the same concentration of 4-n-butylresorcinol for the clinical trial was kindly provided by Enprani (Inchon, Korea). Physical properties of the 4-n-butylresorcinol DMN patch. To validate the morphological properties of microneedles, the 4-n-butylresorcinol DMN patch was visualized using field emission scanning electron microscopy (JSM-7001F, JEOL Ltd., Tokyo, Japan). To evaluate the skin penetration ability of the DMNs, single DMNs were separated from a patch and placed on the stainless steel station of a displacement-force testing machine (Z0.5TN, Zwick/Roell Inc., Ulm, Germany) by attaching double-sided adhesive tape to the


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base of the DMNs. The sensor probe pressed the DMNs at a speed of 3.6 mm/min, and the axial force was recorded. Application of DMN patch to serum-treated pig cadaver skin. Because DMNs dissolve in liquid serum, the appropriate time between serum treatment and DMN patch application was assessed to characterize the optimal conditions for the combined-application method. The time intervals tested were 0, 3, 5, and 7 min, allowing the serum to dry and the DMNs to penetrate serum-treated skin without dissolving. Following the guidelines for cosmetic application (OECD Test Guideline 428: Skin Absorption: In Vitro Method, 2004), 25 µL of serum were applied to hair-removed pig cadaver skin with a surface area of 2.5 cm2 (10 µL per cm2), which was placed on a flat surface. After drying at 37°C and 40% relative humidity using a temperature regulator (Jeio Tech, Seoul, Korea), 5 × 5 arrays of 4-nbutylresorcinol DMN patch was applied to the skin using the force of a thumb for 5 min. After removing the patch from the skin, the perforations were stained with 0.4% trypan blue solution for 10 min and washed with distilled water for analysis of skin penetration.

In vitro skin permeation studies. In vitro permeation studies were conducted using a vertical-type Franz diffusion cell with circular pieces of hairless mouse dorsal skin. The skin tissue was carefully mounted onto the donor compartment of the diffusion cell (surface area: 1.76 cm2). The receptor chamber was filled with 10 mM phosphate-buffered saline (pH 7.4), satisfying the sink condition, and maintained at 37°C using a water jacket. The studies were conducted using three groups of six cells, composed of the combined-application group, DMN-only group, and serum-only group. In the serum-only group, 17.6 µL of serum were applied (10 µL per cm2), containing approximately 60 µg of 4-n-butylresorcinol. In the DMN-only group, DMN patches contained the same amount of 4-n-butylresorcinol, and were applied using the force of a thumb for 5 min and removed after 3 h. In the combined-



application group, the 4-n-butylresorcinol DMN patch was applied 5 min after the serum application and was removed after 3 h. All experiments were carried out with an occluded donor compartment. At pre-determined time points, 0.5 mL of aliquots were withdrawn from the receptor compartment and the withdrawn was replaced by an equal volume of phosphatebuffered saline. The aliquots were analyzed using high-performance liquid chromatography (HPLC), and the amount of lost due to phosphate-buffered saline replacement was calculated in the permeability graph. The cumulative amount of 4-n-butylresorcinol permeated per unit area was plotted as a function of time, and the steady state permeation rate (Jss) was calculate from the slope.

In vitro skin retention studies. The amount of 4-n-butylresorcinol retained in the skin was determined after 3, 6, 12, 24, and 36 h of the skin permeation studies. The skin was washed several times with distilled water and then gently dried using tissue paper. The skin was cut into small pieces using scissors, then homogenized with 50% v/v methanol solution for 5 min and sonicated in a water bath sonicator (Branson Ultrasonics Corp., Danbury, CT, USA) for 30 min at 25°C. The solution was centrifuged for 10 min at 5,000 rpm, and the content of 4-n-butylresorcinol in the supernatant was determined using HPLC. HPLC conditions for 4-n-butylresorcinol. The quantitative determination of 4-nbutylresorcinol was performed using HPLC with a pump (L-7100), autosampler (L-7200), UV detector (L-7400), and data station (LaChrom Elite; Hitachi Ltd., Tokyo, Japan). To separate 4-n-butylresorcinol, a Capcell Pak C18 column (4.6 × 150 mm, 5-µm particle size, Shiseido Ltd., Tokyo, Japan) was used at a flow rate of 1 mL/min. The mobile phase was acetonitrile:water (57:43) with 10 mM NaClO4. The injection volume was 10 µL and the detection wavelength was 280 nm. The intra- and inter-day precision and accuracy for 4-nbutylresorcinol were estimated in triplicate at three different concentrations of 10, 60, and


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100 µg/mL. The calibration curve was based on seven different standard concentrations. Clinical trial: 4-n-butylresorcinol skin depigmenting effect on hyper-pigmented spots. Twenty-three Korean women aged 30 to 60 years who met the inclusion criteria (Supplementary Table 1) were selected to participate in the 8-week clinical trial. All the women had at least two hyper-pigmented spots on their faces, and were instructed to apply 4n-butylresorcinol serum on the hyper-pigmented spots twice a day. Moreover, the combinedapplication group was instructed to wait 5 min and then apply the 4-n-butylresorcinol DMN patch over the serum-treated area. The procedure was repeated every 3 days. The hyperpigmented spots of the combined-application group and the serum-only group were evaluated at baseline and after 4 and 8 weeks. The skin depigmenting effect was assessed by measuring the melanin index with a Mexameter® MX 18 (Courage-Khazaka Electronic, Köln, Germany) and analyzing the images of the hyper-pigmented region using VISIA ver. 5 (Canfield Scientific, Parsippany, NJ, USA) and Image-Pro Plus (Media Cybernetics, Rockville, MD, USA). Adverse reactions during the study were assessed by both subjects and researchers. Subjective adverse reactions included itching, prickling, ticking, burning, stinging, stiffness and tightness, burning in the eyes, and lacrimation. Objective signs included erythema, edema, desquamation, and the appearance of papules. Statistical analysis of clinical trial data was conducted using the SPSS® software program (IBM, Armonk, NY, USA). The Shapiro-Wilk test for normality was used to determine a normal distribution for variables. Analysis of variables for parametric data was conducted using repeated measures ANOVA. If a value was non-parametric, the data were initially compared using the Wilcoxon signed-rank test. Statistical significance was set to p < 0.05. Changes in values were defined using the following equation: Increase or decrease rate (%) = {(value prior to treatment − value after treatment) /



value prior to treatment} × 100. Stability test. The powdered form of 4-n-butylresorcinol, 0.006% w/v 4-nbutylresorcinol solutions, and 4-n-butylresorcinol DMN patches were sealed and wrapped with aluminum foil, then stored up to 30 weeks in 40°C. The contents were measured at predetermined time points using HPLC.


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RESULTS AND DISCUSSION Morphology and fracture force analysis of DMNs. Prior to in vitro and in vivo experiments, a 4-n-butylresorcinol DMN (Figure 1A) and a DMN patch (Figure 1B) were imaged in order to confirm the morphological uniformity of DMNs. The DMNs showed a uniform cone shape and length, with an average length of 190 ± 30 µm and an average tip diameter of 30 ± 4 µm. The recorded mechanical force of a DMN is shown in Figure 1C. The axial load fracture force of a single DMN ranged from 0.38 to 0.46 N per needle, with an average of 0.422 ± 0.029 N (n = 5). Since the minimum fracture force of a DMN tip required for skin penetration is 0.058 N,26 the fabricated DMN patch was considered well-solidified and capable of penetrating the human skin barrier without breaking.



Figure 1. Morphologies and physical properties of 4-n-butylresorcinol DMN patch. (A) Field emission scanning electron microscope image of a DMN (scale bar: 100 µm). (B) 4-nbutylresorcinol-encapsulated DMN arrays (scale bar: 1 mm). (C) After the probe moved and contacted a DMN, the standard force was recorded as the axial of distance; the peak of the graph indicates the fracture force of the DMN.


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Optimization of DMN patch application on serum-treated skin. DMNs, composed of biodegradable matrix, are designed to dissolve in interstitial fluid after skin penetration, and are therefore usually applied to dry skin. After serum treatment, however, DMN application may result in penetration failure because the serum formulation can hydrate the skin and dissolve the DMNs prior to penetration. For successful skin penetration in a combined application, preventing the dissolution of DMNs on the skin surface is essential, and thus, we evaluated the effect of elapsed time between serum treatment and DMN patch application. As shown in Figure 2A, DMN patches were applied between 0 and 7 min after serum application. In the control group, the DMN patch was applied on dry skin without serum and showed no dissolution of DMN structures; complete micro-channel creations stained with trypan blue solution were also observed (white dotted circle). This confirmed that the fabricated DMN patch could penetrate the skin barrier without breaking, as shown with a mechanical force test. In contrast, when serum was applied, DMN structure dissolution increased as the time interval between serum and patch application decreased (from 3-min to 0-min interval); maximum dissolution was observed when the serum and the patch were applied simultaneously (0-min interval). As expected, fewer channel creations were observed as the time interval decreased, because incomplete insertion was expected to occur when DMN structures were partially dissolved in hydrated skin. When DMN patches were applied 5 and 7 min after serum application, however, DMN structures were fully maintained and skin channels matching the whole microneedle array were perfectly produced. As shown in Figure 2B, the ratio of the number of skin perforations over the total number of microneedles (percent penetration) increased as the time interval increased. The percent penetration was only 34.67 ± 14.73% with a 0-min time interval, and increased from 77.33 ± 9.98% to almost 100% as the time interval increased from 3 min to 5 min. Beyond



the 5-min time interval, the penetration level matched that of the control group, indicating that the topical serum dried completely after 5 min. Therefore, the time interval was set as 5 min in subsequent experiments. Because DMNs applied 5 min after the serum treatment could penetrate the skin successfully, DMN structures were expected to dissolve inside the skin upon contact with interstitial fluid. As shown in Figure 2C, DMN structures were gradually dissolved inside the skin over time which resulted in decrease of the height of microneedle from 92.24 ± 7.32% at 0 min, 73.13 ± 3.29% at 20 min, 53.13 ± 10.79% at 40 min to 17.01 ± 11.88% at 1 h. The image of the DMN structure after 40 min (iii) was similar to the image captured for the 3-min time interval in Figure 2A. This indicates again that the partial dissolution of the structures when 3 min elapsed between serum and DMN patch application was due to undried topical formulations. Only trace residue without microneedle shapes was observed 1 h after DMN application on dried serum-treated skin.


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Figure 2. Skin penetration analysis after 4-n-butylresorcinol serum treatment. (A) In the control group (no serum treatment), DMN structures were maintained, and showed complete micro-channel creations (white dotted circle). When the elapsed time between serum and DMN patch application was 0 and 3 min, partial dissolution of DMN structure due to undried serum and imperfect channel creations were observed. After 5 and 7 min, DMN structures were maintained and showed the same channel creations as the control. (B) Percent penetration of 5-min and 7-min intervals was similar to that of the control group, indicating that the appropriate application time for DMN patches on serum-treated skin is 5 min (n = 4). (C) Dissolution of DMN structures following a 5-min serum treatment shown after i) 0 min, ii) 20 min, iii) 40 min, and iv) 1 h. All scale bars: 500 µm.



Distribution analysis of model drugs encapsulated in serum and DMN patches. The distribution of model drugs in the serum formulation and in the DMN patches was analyzed using Alexa Fluor 568 in the serum and Alexa Fluor 488 in the DMNs. Images of the skin surface and cross-sections were analyzed with a fluorescent microscope after 1 h of DMN application on serum-treated skin with a 5-min interval between serum and patch application. As shown in Figure 3A, Alexa Fluor 568 from the serum formulations was observed in the skin surface images, including the DMN insertion site (arrows). The green fluorescence pattern of Alexa Fluor 488 matched the DMN array was also observed on the surface of the skin at the insertion site. This indicates again that the 5-min interval before DMN application was appropriate, and the DMNs penetrated the skin successfully. Furthermore, cross-section images of the skin clearly showed that Alexa Fluor 568 in the serum formulation was delivered into the skin through the channels created by the DMNs (dotted white circle). This suggests that DMNs can both create skin channels and deliver a pre-treated topical formulation into the skin. In contrast to these findings, immediately after DMN application (0-min interval) showed partial dissolution of DMN structures and incomplete skin insertion. Alexa Flour 568 in the serum formulation also covered the skin surface. However, Alexa Fluor 488 in the DMNs did not show the DMN array pattern and localized only on the skin surface; not penetrating the skin (Figure 3B). This strongly implies that DMN structures were dissolved by the serum on the skin surface and could not penetrate the skin successfully. This finding was also confirmed by the cross-section images, which showed Alexa Fluor 488 only on the skin surface, and not within the skin (white dotted circle). Since DMN structures were dissolved by the serum and could not penetrate the skin, Alexa Fluor 568 in serum formulations was not observed within the skin at the insertion site either. The fluorescence


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signals and the merged images indicate that the drugs were delivered from the skin surface via topical serum and DMN application simultaneously.

Figure 3. Distribution images of Alexa Fluor 568 dye in serum and Alexa Fluor 488 dye



loaded into DMN patches. Arrows indicate DMN insertion site and dotted black line in phase contrast of cross-sectioned images indicates DMN. (A) Alexa Fluor 488 DMN array was applied after 5 min of Alexa Fluor 568 serum treatment. After 1 h of DMN application, Alexa Fluor 488 patterns were observed on the surface of pig cadaver skin from the insertion site, indicating that DMN penetrated the skin barrier successfully. Cross-sectioned images of pig cadaver skin show that the Alexa Fluor 568 dye contained in the serum formulation was delivered into the skin with micro-channels created by sequential DMN application (dotted white circle). (B) DMN patch insertion immediately after serum application showed that the Alexa Fluor 488 in the DMN diffused over the skin surface (dotted white circle) along with Alexa Fluor 568 from the serum formulation and remained on the skin surface, indicating that DMN structures were dissolved by the serum and could not penetrate the skin. All scale bars: 500 µm.


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HPLC assay for 4-n-butylresorcinol. The 4-n-butylresorcinol peak was consistently separated, with a retention time of 6.5 min. The seven-point calibration curve was linear in the concentration range of 1 to 100 µg/mL (R2 = 0.9999). The limits of detection and quantitation were 1 and 2.9 µg/mL, respectively. At a concentration of 10, 60, and 100 µg/mL, inter- and intra-day accuracy (percentage deviation) and precision (relative standard deviation) were within 5%.

In vitro skin permeation and retention studies. The delivery of 4-n-butylresorcinol through a combined-application, a DMN-only application, and a serum-only application was analyzed using Franz diffusion cells to measure the transdermal diffusion over time (Figure 4A). After 12 h of application, the combined-application group showed an overall 34.15 ± 5.67 µg per cm2 delivery of 4-n-butylresorcinol in the receptor compartment; this was higher than the delivery in the DMN-only group (24.63 ± 3.99 µg per cm2) and serum-only group (14.09 ± 4.50 µg per cm2). The release profiles in the serum-only and DMN-only groups differed. In the serum-only group, the absorbed 4-n-butylresorcinol gradually increased over time showing J0→12h of 1.17 µg per cm2·h based on the diffusion kinetics of traditional topical formulations. In the first 1 h, especially, the serum-only group showed J0→1h of 5.79 µg per cm2·h while the DMN-only group showed 14.40 µg per cm2·h. This finding can be attributed to the difference in delivery mechanism between serum and DMN application. In serum-only application, 4-n-butylresorcinol diffused out slowly from the skin surface to the receptor compartment. On the other hand, DMNs penetrated the skin and delivered 4-nbutylresorcinol to the epidermis near the receptor, which means penetration is a dominant factor. The combined-application group exhibited a skin permeation pattern similar to that of the DMN-only group, confirming that the DMN patch penetrated the skin successfully



after serum application. The J0→1h in combined-application group was 17.52 µg per cm2·h, which is the highest value comparing with serum-only and DMN-only group. Moreover, flux value after 1 h permeation (J1→12h) in combined application group was also greatest showing 1.51 µg per cm2·h compared with the serum-only group (0.75 µg per cm2·h) and the DMNonly group (0.93 µg per cm2·h), which could deliver 4-n-butylresorcinol efficiently through both DMN and serum formulations simultaneously. In retention studies, the amount of 4-n-butylresorcinol retained in pig cadaver skin was measured in all groups to evaluate accumulation within the skin (Figure 4B). In the DMN-only group, less 4-n-butylresorcinol was retained because much of the drug was delivered to the receptor compartment, compared with the drug quantities delivered in the serum-only group. However, the combined-application group exhibited higher 4-nbutylresorcinol residues than the other groups, as the total amount of delivered 4-nbutylresorcinol was also higher than that of the other groups. Since 4-n-butylresorcinol targets the melanocytes which are found in the stratum basale of the epidermis, the retained amount of 4-n-butylresorcinol within the skin for a long time is helpful to treat hyper-pigmentation. However, accumulation of 4-n-butylresorcinol by cumulative application of the patch may induce cytotoxicity; thus, the days between DMN patch application in the clinical trial was set based on these findings. Levels of residues in all groups gradually decreased over time, and disappeared within 36 h. Therefore, DMN patch application interval was set as 72 h (2 days for metabolizing and clearing the retained amount, and 1 day for rest).


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Figure 4. Cumulative skin permeation and retention of 4-n-butylresorcinol. (A) The combined group delivered the highest amount of 4-n-butylresorcinol to the skin, and showed a release profile similar to that of the DMN-only group, indicating successful skin penetration (mean ± SD, n = 6). (B) After 36 h, the amount of 4-n-butylresorcinol retained in pig cadaver skin did not accumulate in any group (mean ± SD, n = 3).



Clinical trial: Analysis of melanin index and hyper-pigmented region. Treatments were applied based on the combined-application group and serum-only group every 3 days on hyper-pigmented spots of the facial area for 8 weeks. The melanin index decreased in both groups, indicating that skin brightness improved by the effect of 4-n-butylresorcinol (Figure 5A). Moreover, the decrease in melanin index in the combined-application group was greater than in the serum-only group, since the combined-application group could deliver more 4-nbutylresorcinol into the skin. The melanin index in the combined-application group showed a 2.65% and 5.01% improvement at 4 and 8 weeks, respectively, whereas the serum-only group showed only a 1.92% and 3.39% improvement. The hyper-pigmented area showed a similar improvement trend in both groups (Figure 5B). The combined-application group showed a 3.59% and 5.53% improvement at 4 and 8 weeks, respectively, and the serum-only group showed 3.37% and 5.32% improvement. Based on these clinical data, we confirmed that the combined-application group, where skin permeation of 4-n-butylresorcinol was improved, displayed enhanced efficacy compared to the traditional serum-only group. All melanin index and hyper-pigmented area values in both groups improved significantly after treatment (4 and 8 weeks) compared with baseline values prior to treatment (p < 0.05), and pigmented areas gradually disappeared over the course of the study in both groups (Figure 6). All statistical data are shown in Supplementary Tables 2 and 3.


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Figure 5. Analyses of melanin index and hyper-pigmented area. Following eight consecutive weeks of application, both the combined group and serum-only group showed significant improvement in melanin index (A) and hyper-pigmented area (B) compared with baseline values prior to treatment (mean ± SEM, *p < 0.05 compared with baseline values prior to treatment).



Figure 6. Images of skin replicates (hyper-pigmented spot). Spots gradually disappeared after 8 weeks of application in both the combined group (A), and serum-only group (B).

Safety assessment. After 4 weeks, six out of twenty-three subjects showed mild adverse reactions including itching in one subject (objective desquamation), stinging in another, and skin tightness in four subjects, thus received humectants to apply with the test products. After 8 weeks, skin irritation reactions disappeared in these six subjects, and no side effects were observed in the other subjects during the course of the study (Supplementary Table 4). Stability test. The amount of 4-n-butylresorcinol in all formulations (dry powder, solution, and DMN patches) was evaluated over time at 40°C. All formulations retained more than 95% of their contents over 30 weeks of testing. Thus, 4-n-butylresorcinol encapsulated in DMN patches was considered stable (Supplementary Figure 1).


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CONCLUSION In this study, we introduced a novel DMN application combined with a topical serum treatment as a useful alternative to SMN to improve skin permeation efficiency. We found that DMN arrays created channels and delivered the drug encapsulated in pre-treated topical formulations into the skin. Using a skin-depigmenting agent, 4-n-butylresorcinol, we found that this combination method displayed greater efficacy than traditional serum treatment assessing on melanin index and hyper-pigmented region analyses in a human clinical trial. DMNs are composed of biodegradable polymers and can therefore resolve dose limitation and safety concerns; this novel approach can be developed further to optimize transdermal drug delivery.



CONFLICT OF INTEREST The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the R&D program of MSIP/COMPA (2015K000201, Development of minimal pain multi-micro lancets for one-touch-smart diagnostic sensor) and the Seoul R&BD Program (SS100001).

SUPPORTING INFORMATION Supplementary Table 1. Inclusion and exclusion criteria in the clinical trial Supplementary Table 2. Statistical analysis of melanin index by absorption and reflection Supplementary Table 3. Statistical analysis of hyper-pigmented areas Supplementary Table 4. Skin adverse reactions (n = 43) Supplementary Figure 1. Stability test of 4-n-butylresorcinol formulations


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Table of contents graphic 46x35mm (300 x 300 DPI)



Figure 1. Morphologies and physical properties of 4-n-butylresorcinol DMN patch. (A) Field emission scanning electron microscope image of a DMN (scale bar: 100 µm). (B) 4-n-butylresorcinol-encapsulated DMN arrays (scale bar: 1 mm). (C) After the probe moved and contacted a DMN, the standard force was recorded as the axial of distance; the peak of the graph indicates the fracture force of the DMN. 82x199mm (300 x 300 DPI)


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Figure 2. Skin penetration analysis after 4-n-butylresorcinol serum treatment. (A) In the control group (no serum treatment), DMN structures were maintained, and showed complete micro-channel creations (white dotted circle). When the elapsed time between serum and DMN patch application was 0 and 3 min, partial dissolution of DMN structure due to undried serum and imperfect channel creations were observed. After 5 and 7 min, DMN structures were maintained and showed the same channel creations as the control. (B) Percent penetration of 5-min and 7-min intervals was similar to that of the control group, indicating that the appropriate application time for DMN patches on serum-treated skin is 5 min (n = 4). (C) Dissolution of DMN structures following a 5-min serum treatment shown after i) 0 min, ii) 20 min, iii) 40 min, and iv) 1 h. All scale bars: 500 µm. 177x118mm (300 x 300 DPI)



Figure 3. Distribution images of Alexa Fluor 568 dye in serum and Alexa Fluor 488 dye loaded into DMN patches. Arrows indicate DMN insertion site and dotted black line in phase contrast of cross-sectioned images indicates DMN. (A) Alexa Fluor 488 DMN array was applied after 5 min of Alexa Fluor 568 serum treatment. After 1 h of DMN application, Alexa Fluor 488 patterns were observed on the surface of pig cadaver skin from the insertion site, indicating that DMN penetrated the skin barrier successfully. Crosssectioned images of pig cadaver skin show that the Alexa Fluor 568 dye contained in the serum formulation was delivered into the skin with micro-channels created by sequential DMN application (dotted white circle). (B) DMN patch insertion immediately after serum application showed that the Alexa Fluor 488 in the DMN diffused over the skin surface (dotted white circle) along with Alexa Fluor 568 from the serum formulation and remained on the skin surface, indicating that DMN structures were dissolved by the serum and could not penetrate the skin. All scale bars: 500 µm. 177x223mm (300 x 300 DPI)


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Figure 4. Cumulative skin permeation and retention of 4-n-butylresorcinol. (A) The combined group delivered the highest amount of 4-n-butylresorcinol to the skin, and showed a release profile similar to that of the DMN-only group, indicating successful skin penetration (mean ± SD, n = 6). (B) After 36 h, the amount of 4-n-butylresorcinol retained in pig cadaver skin did not accumulate in any group (mean ± SD, n = 3). 138x232mm (600 x 600 DPI)


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Figure 5. Analyses of melanin index and hyper-pigmented area. Following eight consecutive weeks of application, both the combined group and serum-only group showed significant improvement in melanin index (A) and hyper-pigmented area (B) compared with baseline values prior to treatment (mean ± SEM, *p < 0.05 compared with baseline values prior to treatment). 140x237mm (600 x 600 DPI)



Figure 6. Images of skin replicates (hyper-pigmented spot). Spots gradually disappeared after 8 weeks of application in both the combined group (A), and serum-only group (B). 177x123mm (300 x 300 DPI)


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Enhanced transdermal delivery by combined application of dissolving

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