A Semi-Dissolving Microneedle Patch Incorporating TEMPO-Oxidized Bacterial Cellulose Nanofibers for Enhanced Transdermal Delivery

Although dissolving microneedles have garnered considerable attention as transdermal delivery tools, insufficient drug loading remains a challenge owing to their small dimension. Herein, we report a one-step process of synthesizing semi-dissolving microneedle (SDMN) patches that enable effective transdermal drug delivery without loading drugs themselves by introducing TEMPO-oxidized bacterial cellulose nanofibers (TOBCNs), which are well dispersed, while retaining their unique properties in the aqueous phase. The SDMN patch fabricated by the micro-molding of a TOBCN/hydrophilic biopolymer mixture had a two-layer structure comprising a water-soluble needle layer and a TOBCN-containing insoluble backing layer. Moreover, the SDMN patch, which had a hole in the backing layer where TOBCNs are distributed uniformly, could offer novel advantages for the delivery of large quantities of active ingredients. In vitro permeation analysis confirmed that TOBCNs with high water absorption capacity could serve as drug reservoirs. Upon SDMN insertion and the application of drug aqueous solution through the drug inlet hole, the TOBCNs rapidly absorbed the solution and supplied it to the needle layer. Simultaneously, the needle layer dissolved in body fluids and the drug solution to form micro-channels, which enabled the delivery of larger quantities of drugs to the skin compared to that enabled by solution application alone.


Introduction
The stratum corneum (SC), which is the outermost layer of the skin with a thickness of 10 to 15 µm, acts as a strong barrier to transdermal drug delivery systems. Microneedles that are a few hundred micrometers in length can physically pierce the skin with minimal invasion, which enables the simple, painless, and effective delivery of active ingredients into the skin. Their applicability has been widely studied in various fields, such as cosmetics, biomedicines, and vaccines [1][2][3][4][5]. Beginning with a study [6] in which microneedles consisting of silicon were observed to increase the skin permeability of calcein, various solid microneedles (SMNs) fabricated from metal, ceramic, or glass have been reported [7][8][9]. SMNs create transient micro-channels in the skin through which drugs that are applied topically or coated on the needles are delivered. However, if the needles break within the skin, they can remain within the skin for a long time and cause adverse events such as inflammatory responses.
Meanwhile, dissolving microneedles (DMNs), which are primarily composed of water-soluble polymers, are dissolved following skin penetration, and the drug encapsulated within the matrix is released. Compared to SMNs, DMNs offer various advantages, such as ease of production, convenience backing layer of the patch, the solution spreads rapidly throughout the patch due to the presence of TOBCNs, which are distributed evenly in the backing layer. As the needle layer dissolves in body fluids and the aqueous drug solution simultaneously, the drug is readily delivered to the skin via the micro-channels created by the dissolved needles. The effects of transdermal drug delivery using the SDMN patch were visualized using model drugs, and in vitro skin permeation studies using fluorescein and nanocapsulated retinol were conducted as well. The SDMN patches facilitated enhanced transdermal delivery without drug-loading into the patches, and also enabled the delivery of a broad range of drugs while avoiding drug breakdown. ≥ μ μ Step-wise illustration of the transdermal delivery of a drug loaded in topical aqueous solution by a semi-dissolving microneedle (SDMN) patch. (a) The SDMN patch, consisting of a soluble microneedle layer and an insoluble backing layer where TEMPO-oxidized bacterial cellulose nanofibers (TOBCNs) are evenly distributed, is applied to the skin. Sequentially, an aqueous drug solution is injected through the drug inlet hole in the backing layer of the patch. (b) The TOBCNs rapidly absorb the drug solution while the needle layer is dissolved, thereby creating micro-channels. (c) The TOBCNs serve as a drug reservoir that continuously delivers the drug to the skin via the micro-channels created by the dissolved needles.

Preparation of SDMN Patch
We used the micro-molding technique [32], which has been widely utilized to fabricate conventional DMN patch, to fabricate the SDMN patch. Microneedle molds were prepared by casting the PDMS precursor solution over a microneedle array master (LG Electronics, Pyeongtaek, Korea), which contains 1084 pyramidal-shaped microneedles (250 µm height, 125 µm base width, and 400 µm tip-to-tip spacing), followed by curing at 70 • C for 12 h. For the SDMN mixture solution, HA (0.22 g), CMC (0.18 g), trehalose (0.30 g), glycerin (0.23 g) and Aqua Cellulose Solution TM (1.5 wt% TOBCNs, 5.0 g) were mixed with deionized water (4.07 g). The mixture was casted onto the PDMS negative mold, vacuumed for 30 min and dried at 50 • C for 3 h. Then, the SDMN circular patch (1.5 cm in diameter) was attached to a circular hydrocolloid adhesive with a circular hole (1 cm in diameter) in the middle for skin adhesion, then separated from the mold, and a circular hole (0.6 cm in diameter) for the drug inlet was punched at the center of the patch. For comparison, DMN patches without TOBCNs were also fabricated.

Morphological and Mechanical Characterization of SDMN Patch
The morphology of the microneedles was observed using an optical microscope (Leica DM 1000, Leica, Solms, Germany) and a scanning electron microscope (SEM, S-4800, HITACHI, Tokyo, Japan). To validate the skin penetration ability, the prepared SDMN patches were applied on the porcine skins with a force of 20 N for 10 s. After the removal of the SDMN patch, the microchannels created on the skin were selectively stained with trypan blue solution for 1 min, and excess solution was wiped off. The stained microchannels were observed using an optical microscope and compared to the number of needles applied.
The insertion force of the SDMN patch was analyzed by a TA.XT Plus Texture Analyzer (Stable Micro Systems, Surrey, UK). The fresh porcine skin was fixed on a polystyrene foam block that mimics soft tissue under the skin [33] and placed on a platform of the machine. The SDMN patch which was mounted on the cylindrical stainless-steel probe (2.5 cm in diameter) using double-sided adhesive tape (Scotch double-coated tape 665, 3M, St. Paul, MN, USA) was moved towards the porcine skin at a speed of 0.05 mm/s. The loading force and displacement were recorded until the loading force reached 30 N.

Skin Distribution Analysis of Model Drug
The distribution of model drugs loaded in topical aqueous solution was observed using rhodamine B and fluorescein. Firstly, the porcine skin, wiped off with a tissue, was placed on an aluminum foil. Then, a SDMN patch was attached to the skin with a force of 20 N for 10 s, and the skin with the SDMN was carefully mounted on the Franz diffusion cell having a 2.83 cm 2 of diffusion area. The receptor solution was composed of 2.5 mL of PBS buffer (pH 7.4) to achieve sink condition. To confirm the horizontal diffusion of an aqueous drug solution, photographs were taken after 30 min of rhodamine B aqueous solution (300 µg/mL, 100 µL) application. For comparison, the skin with DMN without TOBCNs was used as control. Additionally, to assess the vertical diffusion, fluorescein aqueous solution (50 µg/mL, 2 mL) was applied onto the drug inlet hole in the backing layer of the SDMN patch, which was inserted in the porcine skin, and maintained at 37 • C and 50% relative humidity for 3.5 h. A control experiment was performed with the same fluorescein solution on the porcine skin without the SDMN patch. After the removal of the remaining fluorescein solution and the SDMN patch, the skin cross-sectional images were analyzed via a microscope (EVOS FL Auto 2, Thermo Fisher Scientific, Waltham, MA, USA) to verify vertical diffusion of fluorescein.

In Vitro Skin Permeation Studies
The in vitro permeation study was carried out with the Franz diffusion cell, as described above. The porcine skins and receptor mediums were withdrawn after 3.5 and 24 h of application. To determine the amount of sample absorbed in the SC, tape-stripping was repeated on the same skin area 3 times with a pressure applicator and stripping tapes. Each tape was collected in an individual 1.5 mL Eppendorf tube (Eppendorf, Hamburg, Germany), followed by adding 1 mL of PBS solution to the tube, and maintained for 24 h to sufficiently extract the fluorescein absorbed in the SC. The solutions for the three tapes were analyzed to determine the sample absorbed in SC. Meanwhile, the tape-stripped porcine skin was placed in plastic zipper bag, soaked in distilled water at 70 • C for 30 s, and separated into epidermis and dermis by pushing gently the skin surface with a pusher. Each tissue was homogenized in PBS solution (1 mL) using a Precellys 24 homogenizer (Bertin Technologies, Montigny, France), and centrifuged (Centrifuge 5427R, Eppendorf, Hamburg, Germany) at 13,000 rpm for 5 min. Each supernatant was collected to determine the permeation amount of fluorescein by using a fluorescence spectrometer (Varioskan Lux, Thermo Fisher Scientific, Waltham, MA, USA). The statistical tools within the Microsoft Excel program (Redmond, WA, USA) were used to calculate p values. The p values were calculated by a Student's t-test with equal variance to compare the groups with and without SDMN patch application.
Prior to the in vitro permeation study for retinol, retinol was encapsulated with lipids as the capsule matrix. Briefly, retinol 50C (5 g) was dissolved in Palmester 3580 (2.5 g) and mixed with melted Compritol 888 ATO (3 g) and stearylamine (2 g). The lipid mixture was added dropwise to Tween 60 aqueous solution (10 wt%) at 80 • C under vigorous stirring, resulting in pre-emulsion. The pre-emulsion was further sonicated for 10 min and cooled down to room temperature and stored in a refrigerator before use. The hydrodynamic diameter of the retinol nanocapsules was measured by the dynamic light scattering (Nano-ZS90, Malvern Instruments, Worcestershire, UK). After the insertion of the SDMN patch into the porcine skin, the aqueous retinol nanocapsule dispersion (20.0 µg of retinol) was applied to the drug inlet hole of the SDMN patch and other processes were carried out as described above.

SDMN Preparation and Characterization
The SDMN patch was fabricated using the micro-molding method [32], which has been adopted previously to prepare conventional dissolving microneedles, as shown in Figure 2a (refer to Section 2). Briefly, an aqueous mixture, consisting of 50% Aqua Cellulose Solution TM (containing 1.5% TOBCNs, Figure 2b), 2.2% HA, 1.8% CMC, 3.0% trehalose, 2.3% glycerin, and 40.7% deionized water, was cast on a negative silicone mold. Then, the aqueous mixture-loaded mold was vacuumed and dried at 50 • C for 3 h. After gently detaching the patch from the mold, a circular hole was punched into the backing layer of the patch for drug injection.
Nano-fibrous polymer microneedles with two-layer structures were developed earlier by the two-step casting of a silk fibroin solution and a prepolymer solution for fabricating the needle matrix [34,35]. In contrast, we found that the structural characteristics of TOBCNs enabled the one-step synthesis of two-layer microneedles in which TOBCNs are loaded only in a single layer. We observed that TOBCNs were localized only to the backing layer of the SDMN patch (Figure 2c), which exhibited a uniform distribution of TOBCNs. However, TOBCNs were rarely found on the needle surface and the front surface of the patch ( Figure S1). Due to the small diameter (average diameter of 80 nm) and highly dispersed properties of TOBCNs, they exhibited three-dimensional (3D) and highly entangled network structures, which prevented the packaging of TOBCNs in the microneedle mold cavity. As a result, the TOBCN network structures were located only in the backing layer of the patch, which enabled the synthesis of the two-layer microneedles through single-step casting. To further confirm the dual structure of the SDMN patch, a water droplet (100 µL) was dropped on the SDMN patch (Figure 2d, left), and for comparison, the same procedure was performed using a conventional DMN patch without TOBCNs (Figure 2f). In contrast to the DMN patch, which completely dissolved in water (Figure 2g), the SDMN patch containing the water-insoluble TOBCNs in the backing layer retained its shape after the water droplet evaporated (Figure 2e, left). Meanwhile, the pyramid-shaped microneedle layer (Figure 2d, right) dissolved completely in the water droplet (Figure 2e, right), which indicates that the needle layer did not contain TOBCNs. These results clearly highlight the unique semi-dissolving characteristics of the SDMN patch, where the dissolving needle layer is composed of water-soluble compounds and the water-insoluble backing layer contains TOBCNs. The fabricated SDMN patches are shown in Figure 3a,b. Each patch consisted of 1084 microneedles that had a pyramidal shape with uniform dimensions. The height, base width, and tip-to-tip distance of the microneedles were 250 ± 7, 125 ± 3, and 400 ± 4 µm, respectively. The SDMNs should have adequate mechanical strength to penetrate the SC to deliver active compounds across the skin barrier. To investigate the skin penetration performance of the SDMN, the insertion force during the insertion process of the SDMN into porcine skin was measured (Figure 3c). The resistance force gradually increased with the displacement due to the inherent resistance property of skin after the SDMN contacted the skin. When the insertion force increased with the displacement reached the rupture limit of the porcine skin, the microneedles penetrated the skin at the point "P" shown in Figure 3c. The minimum force required for microneedles penetrating the SC is defined as the penetration force [36,37] which for the SDMN patch comprising 1084 microneedles was approximately 7.2 N. Since the penetration force of a large number of microneedles might be slightly different from each other, the penetration point (P) was certainly broad, rather than clearly observed. The average thumb force that an adult presses the microneedles with is approximately 20 N [38]. Therefore, the SDMN patch had adequate mechanical strength to penetrate the skin with thumb force application. In addition, using a different method, the skin penetration rate of the needle was determined to indirectly evaluate the strength of the needle. As shown in Figure 3d, micro-channels were created with a force of 20 N while attaching the SDMN patch to porcine skin and were dyed. As indicated by the ratio of the number of holes to those of needles, more than 90% of the needles had adequate strength to penetrate the skin.

Distribution Analysis of Model Drugs
To date, several strategies that combine microneedles with drug reservoirs have been developed [39][40][41][42], such as liquid drug reservoir-integrated ceramic porous microneedles [39] or hydrogel-forming microneedle arrays combined with a drug-loaded adhesive backing layer [40,41]. However, in each of these approaches, it is necessary for the microneedles to remain inserted in the skin for a considerable period, which can cause discomfort to the patients and also increase the risk of infection or the chances of the retention of needle residue. In contrast, the SDMN patch, which consists of a dissolving needle layer that does not leave biohazardous sharp waste, and a backing layer containing TOBCNs as a drug reservoir, could provide a high storage capacity for an aqueous drug solution. Accordingly, we investigated whether the backing layer of the SDMN patch, which itself did not contain the drug, could rapidly absorb and retain an externally injected model drug aqueous solution, and simultaneously deliver it to the needle layer. The SDMN patch, the backing layer of which was attached to a hydrocolloid adhesive with a hole (1 cm in diameter) for skin adhesion, was applied to porcine skin, and an aqueous rhodamine B solution was used as a model drug and was subsequently injected into the drug inlet hole (0.6 cm in diameter) in the backing layer. The hole in the backing layer plays an important role in the immediate introduction of the aqueous drug solution between the backing and needle layers. As shown in Figure 4a, the rhodamine B aqueous solution was spread evenly over the backing layer region where the SDMN patch was attached. The drug solution was absorbed directly into the backing layer and did not flow outward, which would be convenient for the user. Even though the DMN patch without TOBCNs had the same hole structure as the SDMN patch, only a part of the injected drug solution was introduced and distributed non-uniformly on the backing layer, whereas the rest formed a droplet on the skin (Figure 4b). The results clearly demonstrate that the TOBCNs, which had high water absorption and retention capacity, were uniformly distributed in the patch backing layer, such that the aqueous drug solution could horizontally diffuse throughout the patch along the TOBCNs.
The vertical distribution of the drug in the skin after DMN patch application was also analyzed to confirm transdermal delivery through the patch. Figure 4c,d show the section of skin after the insertion of the SDMN patch, followed by the application of fluorescein aqueous solution for 3.5 h. Due to contact with body fluids and the injected drug solution, the needle layer was dissolved, which led to the formation of micro-channels in the skin that functioned as pathways for rapid drug delivery. When compared to the image of the application of the drug solution alone (Figure 4e,f), the strong fluorescent green color of fluorescein, which was deeply distributed in the skin, was observed in this image. Meanwhile, when the fluorescein solution was applied without the attachment of the SDMN patch, weak fluorescence was observed only in the SC layer. Taken together, these results indicate that the SDMN patches were able to deliver drugs horizontally and vertically, without taking up the drugs themselves.

In Vitro Skin Permeation Studies
The effect of the SDMN patches on the skin permeation of active compounds was quantitatively evaluated using the Franz cell technique. In the in vitro permeation studies, the SDMN patch was attached to porcine skin composed of SC, epidermis, and upper dermis, and was then carefully mounted on a Franz diffusion cell; this represented the SDMN-combined group. Subsequently, fluorescein aqueous solution was used as a model drug, and its permeation over time was evaluated (Figure 5a). In a comparative study, only aqueous fluorescein solution was applied without SDMN patch application; this represented the control group.
After 3.5 h of application, 711 ± 57 ng/cm 2 of fluorescein was delivered into the skin and the receptor in the SDMN-combined group, while 135 ± 4 ng/cm 2 of fluorescein was delivered in the control group (solution-only treatment). Furthermore, after 24 h of application, the SDMN-combined group showed 1336 ± 66 ng/cm 2 of fluorescein delivery, which was 5.8 times higher than that in the control group (230 ± 24 ng/cm 2 ). Although the same concentration and quantity of fluorescein aqueous solution were used, the permeation rates differed significantly between the two groups; the initial permeation rate of the SDMN group for 3.5 h was significantly greater than that of the control group. In other words, passive diffusion through the skin at a relatively slow rate occurred in the control group, whereas the SC was physically penetrated in the SDMN group, which ensured the rapid delivery of active ingredients into deep layers of the skin. This indicated that the SDMN patch could successfully deliver fluorescein even though the microneedle patch did not contain the compound.
We further analyzed the relative distributions of fluorescein in the porcine skins from the two groups after 24 h of application (Figure 5b). In the control group, there were traces of fluorescein in the dermis (13.1 ± 7.4 ng/cm 2 ) and receptor (4.6 ± 2.8 ng/cm 2 ), and no significant change was observed compared to the values at 3.5 h (9.5 ± 4.7 and 0.3 ± 0.2 ng/cm 2 for the dermis and receptor, respectively; refer to Figure S2). In contrast, the SDMN-combined group showed a further increase in the quantity of fluorescein that permeated into the dermis and receptor (from 34.5 ± 19.8 and 2.8 ± 0.4 ng/cm 2 at 3.5 h to 142.2 ± 24.6 and 23.4 ± 9.8 ng/cm 2 at 24 h for the dermis and receptor, respectively). Although the majority of permeated fluorescein was present in the SC and epidermis (88% of total permeated fluorescein), due to the relatively short height of the microneedle (~250 µm) compared to the skin thickness, the results clearly indicate that as a skin penetration enhancer, the SDMN patch could effectively deliver large quantities of relatively small water-soluble molecules. To further demonstrate the versatility of the SDMN patches, we attempted to deliver retinol nanocapsules, which are significantly larger than fluorescein. Retinol, a lipophilic vitamin, promotes epidermal cell proliferation, which reduces skin wrinkling and an uneven tone [43]. However, since retinol is chemically unstable and readily oxidized, several retinol encapsulation methods have been developed with an aim to improve its stability [44][45][46][47]. Moreover, DMN patches are primarily composed of hydrophilic polymers and require a high-temperature drying process, which further complicates the process of the homogeneous loading of lipophilic retinol while maintaining its bioavailability. Even if retinol is loaded in an encapsulated form, its efficacy may not be ensured due to the limited loading efficiency of the capsules and DMNs, respectively [11]. Herein, we demonstrated that the SDMN patch could effectively deliver a sufficient level of nanocapsulated retinol even when retinol was not contained in the needles.
Retinol was first encapsulated in a lipid matrix to facilitate chemical stabilization and water dispersion; the hydrodynamic diameter of the capsules was 349 nm (refer to Section 2). After the application of the SDMN patch, the retinol capsule-loaded aqueous solution was applied to the drug inlet hole for 17 h. As shown in Figure 6, the SDMN-combined group showed a permeation of 9.2 ± 0.3 µg/cm 2 , which was higher than that in the control group (4.5 ± 0.6 µg/cm 2 ). In particular, when applied using the SDMN patch, the quantity of retinol delivered under the SC was clearly enhanced compared to that observed in the control group. It was reported that 1.4 µg of retinoic acid-loaded microneedle patch (area of 0.8 cm 2 ) application induced epidermal cell proliferation and significantly accelerated the SC turnover [48]. Since retinol exhibited more than half the magnitude of the efficacy of retinoic acid at the same concentration [49], the level of retinol delivered by the SDMN patch has the potential to give the skin sufficient efficacy. In other words, the SDMN patch enables the delivery of small hydrophilic drugs as well as nanocapsulated lipophilic drugs with improved delivery efficiency and bioavailability. * Figure 6. Retinol content in different skin compartments (SC, epidermis, dermis, and receptor) after 17 h of retinol nanocapsule application in the absence or presence of the SDMN patch. Mean ± SD, n = 2. * p < 0.05, statistically significant difference between the control group (without SDMN) and the SDMN-combined group.

Conclusions
TOBCNs owing to their excellent water affinity and the ability to retain their fiber structure in the aqueous phase, were successfully introduced into DMNs, and imparted a semi-dissolving characteristic. Using the structural characteristics of TOBCNs, a two-layer SDMN patch consisting of a soluble needle layer and an insoluble backing layer containing TOBCNs was fabricated in a one-step process, without the conventional two-step casting process. Upon the injection of the drug in the aqueous solution into the inlet hole in the backing layer, the TOBCNs served as a reservoir that quickly absorbed and continuously supplied the solution into the needle layer. The drug could be delivered through the micro-channels created after the needles dissolved in the aqueous solution and body fluids. In the in vitro skin permeation study, compared to the application of only the solution, the application of the SDMN patch facilitated the delivery of a high quantity of drugs without drug loading in the patch. These results are expected to be useful in the field of cosmetics and biomedical research in the future.

Conflicts of Interest:
The authors declare no conflict of interest.