Skin permeability and magnetic susceptibility analysis of hyaluronic acid-based magnetized microneedles containing iron oxide nanoparticles

Iron oxide nanoparticles (NPs) are biocompatible, have excellent magnetic properties, respond to external magnetic fields, and have been used in many studies on drug delivery systems. In this study, hyaluronic acid (HA)–superparamagnetic microneedles containing superparamagnetic iron oxide nanoparticles were manufactured using HA as the matrix material. Optical and scanning electron microscopy (SEM) were used to investigate the characteristics of the magnetized microneedles (MMNs). The mechanical rigidity of MMNs for in vivo evaluation is expected to be sufficient, as evidenced by a transmittance greater than 90%. Additionally, the superconducting quantum interference device-vibrating sample magnetometer measurement results confirmed that the magnetic characteristics did not change, even after the magnetic nanoparticles (MNPs) were manufactured. The hysteresis curves of the NPs confirmed the maintenance of the magnetic properties, including a coercivity of 60 Oe and susceptibility of 4 × 10 -6 emu/gOe. Based on these results, MNPs are expected to be useful as a delivery material for transdermal drug delivery systems, magnetic resonance imaging, and molecular imaging.


I. INTRODUCTION
Nanoparticles (NPs) are particles ranging in size from 0.1 nm to 100 nm that exhibit physicochemical properties entirely distinct from those of their bulk counterparts.NPs have been extensively explored for diagnostic and therapeutic applications.When NPs are used as contrast agents for diagnostic purposes, the specificity and sensitivity of imaging can be significantly improved.2][3] The expanding scope of NP-technology applications has bolstered research interest in this area, and many studies have been conducted for various purposes.Iron oxide magnetic nanoparticles (MNPs) exhibit magnetic properties, the ability to respond to external magnetic fields, and excellent biocompatibility.5][6] MNPs have been combined with magnetic resonance imaging (MRI).Furthermore, their efficacy as a material for research in the field of diagnostic medicine, for example as MRI contrast agents, has been investigated.In particular, among iron oxide NPs, superparamagnetic iron oxide nanoparticles (SIONs) are widely used to develop various biomedical applications because they are easy to move to a target area using an external magnetic field. 7,8icroneedles (MNs) are transdermal drug delivery systems (TDDS) that pierce fine physical holes in the skin's stratum corneum with micro-sized needles, effectively delivering active drugs using these delicate passages.Recently, various studies have been conducted on the delivery of active ingredients using MN technology in a way that can overcome the limitations TDDS face regarding active ingredient delivery by the stratum corneum. 9Although the injection delivery method offers excellent efficiency in the delivery of the active drug, it requires professional medical technology making self-administration difficult, and poses the risk of needle pain and infection when administered. 10,11Compared with these methods, the MN delivery method is a technology that allows for minimally invasive delivery of active drugs through the skin, eliminating the need for relatively painful needles.
In this study, hyaluronic acid (HA), a biodegradable polymer, was used as an excipient, which is the matrix of MNs, to manufacture HA-MMNs (hyaluronic acid-based magnetized microneedles) containing SIONs in the matrix. 12The mechanical properties were confirmed by measuring the surface shape of the prepared HA-MMNs and the skin permeability intensity of the MMNs through biomembrane (porcine skin).

II. MATERIAL AND METHODS
Absolute Mag TM Amine Magnetic Nanoparticles (Dextran Coated, 50 nm) and bio sodium hyaluronate (molecular weight 1.3-1.8MDa) were used for this study.The master for the MNs was designed with an aspect ratio of 2:1 with a height of 500 μm and a width of 250 μm.A circular disk was fabricated, measuring 15 mm in outer diameter, with the needle area having an outer diameter of 12 mm, and incorporating an MN patch of 750 μm.To manufacture the MNs' elastic mold, a polydimethylsiloxane (PDMS) elastic mold solution was prepared by mixing a content ratio of AK 35 C to 30% of the total of A and B. The three reagents used, ELASTOSIL ® RT 623 A, ELASTOSIL ® RT 623 B, and AK 35 C were maintained at a fixed ration of A:B = 90:10.The needle disk was baked until it was completely saturated with the PDMS elastic mold solution, degassed in a vacuum chamber (550 torr/30 min), and solidified for more than 48 h at room temperature.A flexible elastic mold was produced by removing the solidified mold solution from the needle disk.Next, 0.8 g of the solution that was prepared for the production of HA-MMNs containing SIONs was applied to the mold surface to be used for the needle structure of the PDMS elastic mold.Following the even distribution of the applied solution, air bubbles were removed using a centrifuge (1000 rpm/5 min) and vacuum chamber (550 mmHg/20 min).The solution-filled elastic mold was dried for 48 h in a desiccator, maintaining a humidity level of 30%.Finally, the PDMS elastic mold and the molded HA-MMNs were separated to produce a sample with the same morphology as the needle disk.
The morphologies of the manufactured MNs were evaluated using a digital optical microscope and scanning electron microscope (SEM).As the manufactured HA-MMNs must be inserted through the stratum corneum of the skin, certain very specific mechanical properties are required.These were confirmed by measuring the skin permeability intensity of the polymer artificial membrane (Strat-M TM Membrane, Millipore) and the biomembrane of porcine back skin (Micropig ® Franz Cell Membrane, Apures Co., LTD, Gyeonggi-do, Korea).After forced insertion of MNs into the polymer membrane and porcine skin, trypan blue was added, and the samples were incubated for 5 min.Images were captured following the removal of the trypan blue using a cotton swab.Porcine back skin was used to analyze the magnetization characteristics of the MNs.The MNs were inserted at a force of 50 N for 10 s.After their removal, the magnetization properties of the skin at the insertion sites were tested.Magnetic hysteresis (M-H) curves for two distinct sets of HA-MMNs applied to two separate patches of porcine back skin were measured using a superconducting quantum interference device vibrating sample magnetometer (SQUID-VSM) from Quantum Design (San Diego, CA, USA) to analyze the magnetization characteristics.

III. RESULTS AND DISCUSSION
The SIONs used in this study were nanocomposite products (San Diego, USA) and consisted of a dextran outer shell with an amine group attached to the surface of each MNP. 13 The preparation of MNs during the manufacturing process can result in surface and top shock damage to the MMNs owing to the aggregation and desorption of SIONs. 12Optical digital microscope (Taiwan RoHS) images of the surface and morphology of the SION-containing HA-MMNs can be seen in Fig. 1a.Further characterization achieved using SEM can be seen in Fig. 1b.The manufacturing process had verifiably not resulted in any changes caused by active ingredients to the morphology or surface state.Figure 1 shows the detailed morphologies and SEM images of the prepared HA-MMNs by magnification level.
In addition, mechanical strength was confirmed by measuring the skin permeability of the MMNs using both a polymer artificial membrane and porcine back skin.After confirming the permeability of the polymer synthetic film, porcine back skin (20 mm × 20 mm, 1.2 mm T) stored in the freezer (-20 ○ C) was thawed at room temperature, allowing it to absorb surface moisture.The skin was stretched from each corner to achieve suitable tautness to be comparable to actual skin.The MMNs array was placed on both the membrane and skin, and pressurized for 10 s with a force of 50 N.After removing the MMNs, 1 mL of trypan blue solution was added and incubated for 10 min at room temperature.The number of permeations was compared with the total number of needles.The polymer artificial film was 100% permeable, as shown in Fig. 2a.It can be concluded that MMNs exhibit ample mechanical strength to penetrate both the surface of the polymer membrane and its porous internal structure.The permeability of biofilms was further validated using commercially purchased porcine skin.The MMNs were pressurized (50 N for 10 s) and removed from the porcine back skin.The holes caused by their penetration were stained using the dyeing reagent trypan blue 0.4% (w/w) to determine the number of penetrations.As shown in Fig. 2b, the mechanical rigidity of the MMNs is expected to be sufficient for in vivo evaluation, as evidenced by a transmittance of more than 90%.Variations in the permeability of the MMNs through the polymer artificial film and the porcine back skin may be attributed to differences in the initial pressure applied per unit surface area, influenced by the moisture state of the membrane surface and structural variations within the membrane.
A SQUID-VSM was used to analyze the supermagnetic properties of the MNPs.The magnetic sensitivity of the VSM was 10 -9 emu and set to a measurement range of ±7 Tesla.The magnetic properties of the MNs were evaluated using hysteresis analysis.The magnetization properties of the produced HA-MMNs, which contained MNPs at a concentration of 1 mg/mL, and the HA-MMNs patch-applied to porcine back skin were analyzed independently.The change in magnetic susceptibility was confirmed based on the MNPs' concentration ratio.Figure 3 verifies that the magnetic characteristics, as evidenced by the M-H curve of the MNPs, remain consistent even after the manufacture of HA-MMNs.The M-H curves retain the same magnetic properties, having a coercivity (Hc) of 60 Oe and a susceptibility (χ) of 4 × 10 -6 emu/gOe.The saturation magnetization value of the MNPs in the porcine back skin used in this study was 2 × 10 -4 emu/g at a magnetic field of 1 kOe, and the magnetization value was approximately 1.8 × 10 -4 emu/g at a magnetic field of 600 Oe.We confirmed that the magnetization properties were retained after the MMNs were fabricated and stamped onto the porcine back skin.

IV. CONCLUSION
HA-MMNs containing SIONs were manufactured, the surface and morphology were evaluated, and the permeability of MMNs through polymer synthetic and porcine back skin was analyzed.During the MMNs' manufacturing process, no significant changes in the formulation morphology or surface state were observed.Permeability test results confirmed that the properties were maintained at 90% or higher, indicating that MMNs have sufficient mechanical strength for skin penetration.Moreover, HA-MMNs were proven to be sufficiently stable for in vivo use because their magnetic properties are not deformed during the manufacturing process, preserving their superparamagnetic properties.This information is useful for determining the mixing ratio of iron oxide and hyaluronic acid in evaluating the efficacy of HA-MMNs, as well as for refining the formulation process of products.Furthermore, HA-MMNs can be confidently applied in vivo, contributing to the advancement of both simultaneous molecular imaging and therapy.

FIG. 3 .
FIG. 3. The M-H curves of HA-MMNs patch and porcine back skin.The left image panels show nanoparticle stock solution, microneedles, and porcine back skin forced with the microneedles.The right image presents the M-H curves of HA-MMNs and porcine skin.The inset image shows the M-H curve of porcine skin at a smaller scale.Here, the values of coercivity (Hc) and susceptibility (χ) that can be found from the two M-H curves are 60 Oe and 4 × 10 -6 emu/g Oe, respectively.