**Transdermal Composite Microneedle Composed of Mesoporous Iron Oxide Nanoraspberry and PVA for Androgenetic Alopecia Treatment**

**Jen-Hung Fang <sup>1</sup> , Che-Hau Liu <sup>1</sup> , Ru-Siou Hsu <sup>1</sup> , Yin-Yu Chen <sup>1</sup> , Wen-Hsuan Chiang <sup>2</sup> , Hui-Min David Wang <sup>3</sup> and Shang-Hsiu Hu 1,\***


Received: 29 May 2020; Accepted: 19 June 2020; Published: 22 June 2020

**Abstract:** The transdermal delivery of therapeutic agents amplifying a local concentration of active molecules have received considerable attention in wide biomedical applications, especially in vaccine development and medical beauty. Unlike oral or subcutaneous injections, this approach can not only avoid the loss of efficacy of oral drugs due to the liver's first-pass effect but also reduce the risk of infection by subcutaneous injection. In this study, a magneto-responsive transdermal composite microneedle (MNs) with a mesoporous iron oxide nanoraspberry (MIO), that can improve the drug delivery efficiency, was fabricated by using a 3D printing-molding method. With loading of Minoxidil (Mx, a medication commonly used to slow the progression of hair loss and speed the process of hair regrowth), MNs can break the barrier of the stratum corneum through the puncture ability, and control the delivery dose for treating androgenetic alopecia (AGA). By 3D printing process, the sizes and morphologies of MNs is able to be, easily, architected. The MIOs were embedded into the tip of MNs which can deliver Mx as well as generate mild heating for hair growth, which is potentially attributed by the expansion of hair follicle and drug penetration. Compared to the mice without any treatments, the hair density of mice exhibited an 800% improvement after being treated by MNs with MF at 10-days post-treatment.

**Keywords:** 3D printing process; mesoporous iron oxide; microneedles; minoxidil

### **1. Introduction**

For male baldness, this genetic disease occurs at age 30–60, which is estimated that over 50% of the general population suffer from hair loss [1]. Traditionally in the treatment of hereditary male baldness, the drug was mixed into the inactive component which can soften the stratum corneum, such as glycerol and propylene glycol, facilitating the active ingredient effective for percutaneous absorption [2]. However, the stratum corneum softening takes a long time, with side effects including redness, swelling, rash, and subjective feeling of burning sensation was caused in some users. Additionally, it may lead to hair growth on the undesired local with inaccurate using [3].

As a new type of painless administration, microneedles (MNs) with the advantages of convenient use, minimally invasive procedure, and relatively inexpensive cost, are easy to overcome stratum corneum to facilitate pharmaceuticals entering epidermal as well as dermal layers [4]. MNs are applied in many kinds of researches nowadays. For example, Li et al., last year, designed biodegradable MNs with an air bubble inside the center for rapidly separating chemicals to skin and caused sustained release [5]. However, the conventional fabrications of MNs are rigid, expensive, and inconvenient by directly purchasing commercial products, and using laser ablation, photolithograph, and magneto liquid forming, which prodigiously limits the applications of MNs [6–12]. are applied in many kinds of researches nowadays. For example, Li et al., last year, designed biodegradable MNs with an air bubble inside the center for rapidly separating chemicals to skin and caused sustained release [5]. However, the conventional fabrications of MNs are rigid, expensive, and inconvenient by directly purchasing commercial products, and using laser ablation, photolithograph, and magneto liquid forming, which prodigiously limits the applications of MNs [6– 12].

Magnetic mesoporous nanoparticles consisted of silica as well as iron oxide, and are broadly employed in many fields such as catalyst, cancer therapy, antibacterial composites, energy storage, and drug delivery system [13–19]. These nano-systems provide promising and novel approaches for improving the efficacy with their large pore surface, which can also be easily controlled by adjusting the composition and concentration of surfactant. Especially, iron oxide nanoparticles allow them to be manipulated with externally applied magnetic field, which possess on-demand release behaviors from their cargos without any lag in response. By means of employing magnetic field, iron oxide can slightly generate heat to cause cutaneous blood flow increases as well as vasodilation, leading to the promotion of hair growth [20–22]. Furthermore, iron is one of the most important nutritional factors in human body, as it can help oxygen delivery as well as carbon dioxide removal from animal tissue, and has the composition of serum ferritin [23]. Many studies also show that iron-deficiency is also common with hair loss [24,25]. Magnetic mesoporous nanoparticles consisted of silica as well as iron oxide, and are broadly employed in many fields such as catalyst, cancer therapy, antibacterial composites, energy storage, and drug delivery system [13–19]. These nano-systems provide promising and novel approaches for improving the efficacy with their large pore surface, which can also be easily controlled by adjusting the composition and concentration of surfactant. Especially, iron oxide nanoparticles allow them to be manipulated with externally applied magnetic field, which possess on-demand release behaviors from their cargos without any lag in response. By means of employing magnetic field, iron oxide can slightly generate heat to cause cutaneous blood flow increases as well as vasodilation, leading to the promotion of hair growth [20–22]. Furthermore, iron is one of the most important nutritional factors in human body, as it can help oxygen delivery as well as carbon dioxide removal from animal tissue, and has the composition of serum ferritin [23]. Many studies also show that iron-deficiency is also common with hair loss [24,25].

In this study, the concept of a physical promotion method for patching increases the efficiency of percutaneous absorption and reduces the chance of inducing side effects. A highly biocompatible polyvinyl alcohol was used to make a dissolvable microneedle patch, reaching the smart device in biomedical applications. Digital light processing (DLP) 3D printing technology was used to fabricate the MNs master with high resolution, inexpensive cost, flexible manufacturing, customization, and fast forming prototype compared with conventional process [26]. A mesoporous iron oxide nanoraspberry (MIO) inside MNs (MIOs@MNs) encapsulated Minoxdil (Mx), a therapeutic drug for hair regrowth, which can be triggered by external magnetic field (MF), resulting in local temperature increases as well as controlled release (Figure 1). We further evaluated the biocompatibility and mechanical properties of the MNs, suggesting a potential application in biomedical engineering filed. In this study, the concept of a physical promotion method for patching increases the efficiency of percutaneous absorption and reduces the chance of inducing side effects. A highly biocompatible polyvinyl alcohol was used to make a dissolvable microneedle patch, reaching the smart device in biomedical applications. Digital light processing (DLP) 3D printing technology was used to fabricate the MNs master with high resolution, inexpensive cost, flexible manufacturing, customization, and fast forming prototype compared with conventional process [26]. A mesoporous iron oxide nanoraspberry (MIO) inside MNs (MIOs@MNs) encapsulated Minoxdil (Mx), a therapeutic drug for hair regrowth, which can be triggered by external magnetic field (MF), resulting in local temperature increases as well as controlled release (Figure 1). We further evaluated the biocompatibility and mechanical properties of the MNs, suggesting a potential application in biomedical engineering filed.

**Figure 1.** Schematic mechanism of microneedle patch system applying on the surface of skin. Polyvinyl alcohol (PVA) was used to prepare microneedles (MNs) with dissolvable properties. A mesoporous iron oxide nanoraspberry (MIO) encapsulating Minoxdil (Mx) inside the MNs were triggered by external magnetic field (MF), leading to local temperature increases as well as controlled **Figure 1.** Schematic mechanism of microneedle patch system applying on the surface of skin. Polyvinyl alcohol (PVA) was used to prepare microneedles (MNs) with dissolvable properties. A mesoporous iron oxide nanoraspberry (MIO) encapsulating Minoxdil (Mx) inside the MNs were triggered by external magnetic field (MF), leading to local temperature increases as well as controlled release.

#### release. **2. Materials and Methods**

### *2.1. Materials*

FeCl3·6H2O, ethylene glycol, sodium acetate (NaAc), oleylamine, Mx, and phosphate buffered saline were purchased from Sigma-Aldrich, St. Louis, Missouri, USA. CdSe/ZnS core/shell Quantum Dot Solid (QDs) were purchased form Ocean Nano Tech, San Diego, California, USA. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT reagent) were bought from Amresco, Solon, Ohio, USA. Ham's F-12 medium, fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gibco, Thermo Fisher Scientific, Waltham, Massachusetts, USA. Trypsin (2.21 mM EDTA Solution) was purchased from Corning, Steuben County, New York, USA. C57BL/6 mice obtained from National Laboratory Animal Center, NLAC, Taiwan.

### *2.2. Synthesis of MIOs*

The synthesis process of MIOs follows the hydrothermal method of our previous studies [27]. Briefly, 5.4 g of FeCl3·6H2O were dissolved in 40 mL of ethylene glycol with vigorous stirring till the color of the solution changed to a clear yellow. Then, anhydrous NaAc (3.29 g) was added to the mixture in gentle stirring thereafter to the extent of homogeneity overnight. After the color of the mixtures changed to black, 0.6 mL of oleylamine was added under ultrasonication for effective mixing. Subsequently, 20 mL of the solution was transferred into a teflon-lined stainless steel autoclave (25 mL capacity) for hydrothermal reactions at 220 ◦C for 5, 15, and 25 h, respectively. After the Teflon-lined stainless steel autoclave was allowed to cool down to room temperature, the precipitate was washed by ethanol, three times, and stored in 4 ◦C, this was called MIOs. MIOs were dried under vacuum at room temperature before characterization and applications later. For Mx encapsulation, Mx was loaded to MIOs-5h by mixing 1% Mx with MIOs in dimethyl sulfoxide (DMSO) for 12 h. Then, the mixture was placed into vacuum at 50 ◦C for 24 h to evaporate DMSO. The drug concentration was determined by ultraviolet–visible (UV-vis) spectroscopy (Metertech, Taipei, Taiwan).

#### *2.3. Fabrication Process of MNs*

We used 3D printing technologies to prepare MNs master so that we can freely adjust the specification of MNs including height, width, and length of MNs. Each needle was 300 µm in a round base diameter and 600 µm in height. Needles were arranged with 600 µm tip-tip spacing. Polydimethylsiloxane (PDMS) was placed over the 3D printed MNs master to make female mold, followed by curing in the oven at 60 ◦C for 8 h, and the female mold was finally finished by carefully peeling the 3D printed MNs. For preparation polyvinyl alcohol (PVA)-MNs patch, the mixture, PVA and Mx-MIOs, were loaded onto PDMS female mold, and placed in a centrifuge at 4500 rpm for 1 h, so that the mixed solution would fill the pyramidal cavity of the mold. The back of MNs was filled with pure PVA solution, and centrifuged at 4500 rpm and 25 ◦C for 1 h. Subsequently, the device was allowed to dry at room temperature for 12 h, and then the patch was carefully detached from PDMS mold.
