A computer-aided design (CAD) program (DeskArtes Oy, Espoo, Finland) was used to prepare an STL format file for fabrication of the microneedle array master structure. The master structure was a 5 × 5 array of 25 identical solid microneedles (needle height = 500 µm; needle base diameter = 150 µm; needle center to needle center distance = 500 µm).
The 2PP technique was used to create a microneedle master.14,16,22
This process involves nonlinear light absorption, igniting chemical reactions and material hardening within well-defined, highly localized volumes. The 2PP process involves spatial and temporal overlap of photons in order to bring about chemical reactions, which lead to photopolymerization and material hardening within well-defined, highly localized volumes. A titanium: sapphire laser (Kapteyn-Murnane, Boulder, CO) was used to obtain femtosecond laser pulses (60 fs; 94 MHz; <450 mW; 780 nm), which were focused using a 10× plan achromat microscope objective. Nonlinear absorption of laser pulses cleaved chemical bonds of photoinitiator molecules located in a small focal volume within the polymer resin. The radicalized photoinitiator molecules created radicalized polymolecules through interaction with the monomers; reactions were terminated when radicalized polymolecules interacted with other radicalized polymolecules. By moving the laser focus in three dimensions within the photopolymer, material was polymerized along the laser trace. A combination of three C-843 linear translational stages (Physik Instrumente, Karlsruhe, Germany) and a galvo scanner (Scanlab AG, Puchheim, Germany) was utilized in order to alter the laser focus position in three dimensions.
The original microneedle array was fabricated by 2PP of SR 259 polymer (Sartomer, Paris, France) containing 2 wt % Irgacure 369 photoinitiator (Ciba Specialty Chemicals, Basel, Switzerland) on a glass cover slip. SR 259 is a polyethylene glycol (200) diacrylate that exhibits low volatility, a refractive index of 1.4639, viscosity of 25 cps at 25 °C, surface tension of 41.3 dynes/cm, and a molecular weight of 302.23
369 photoinitiator exhibits an absorption peak at ~λ = 320 nm. The microneedle array was subsequently sputter coated with gold (coating time = 245 s; coating current = 10 mA) to improve separation of the master structure from the mold. The glass cover slip containing the microneedle array was then fixed to a glass microscope slide using double-sided tape.
A negative mold of the microneedle array was fabricated using PDMS. Sylgard® 184 silicone elastomer and hardening agent (Dow Corning, Midland, MI) were prepared according to manufacturer instructions. The liquid mixture was subsequently degassed under vacuum. After degassing, a 20 mm diameter open center aluminum crimp seal (Sigma Aldrich, St. Louis, MO) was placed on the glass with the microneedle array located in the center of the ring. The unpolymerized silicone elastomer was poured over the microneedle array until the aluminum ring was completely filled. The glass slide with microneedle array, aluminum ring, and silicone elastomer were placed under 100 mbar vacuum in order to remove residual gas voids. The sample, consisting of a glass slide, microneedle array, aluminum seal, and silicone, was subsequently placed on a hot plate for PDMS cross linking. The hot plate temperature was increased from 25 to 100 °C over 30 min and then maintained at 100 °C for 30 min. Once the polymerized mold had cooled, it was mounted onto a C843 linear translational stage (Physik Instrumente, Karlsruhe, Germany) in order to separate the mold from the master structure. The glass slide was held in place against the table using two metal restraining bars, while the aluminum seal containing the PDMS mold was vertically moved by the stage. A second PDMS mold (depth = 2 mm; diameter = 1 cm) was used for molding the substrate.
Approximately 50 µl of eShell 200 polymer (Envisiontec, Gladbeck, Germany) was placed on the surface of the PDMS mold over the microneedle array. The mold containing eShell 200 polymer was subsequently degassed under vacuum in order to allow the eShell 200 polymer to completely fill the mold. The cylindrical substrate mold was then filled with eShell 200 polymer, and the microneedle mold was placed on top of it. The two molds were placed in contact, with the substrate mold facing up and the microneedle mold facing down so that the two surfaces with eShell 200 were in contact. The molds were then exposed for 2 min to an ultraviolet curing lamp, which provided visible and ultraviolet light emission (Thorlabs, Newton, NJ). The molds were subsequently inverted; the substrate side was exposed to ultraviolet light for 1 min. After ultraviolet curing, the molds and polymerized microneedle arrays were separated by hand.
Images of the eShell 200 microneedles were obtained using a S3200 scanning electron microscope (Hitachi, Tokyo, Japan), which was equipped with a Robinson back-scattered electron detector. Energy dispersive x-ray spectroscopy was performed to determine the chemical composition of the microneedles. Compression testing of the eShell 200 microneedle arrays was performed using an Electroforce 3100 system (Bose, Eden Prairie, MN). The eShell 200 microneedle arrays were placed directly on the load cell surface. Axial loading was applied to the microneedle array (displacement rate = 10 µm/s) until a force of 10 N was achieved. Compression testing of microneedles against hard surfaces such as polytetrafluoroethylene has been used to examine the mechanical properties of microneedle arrays.14,16,17
Human epidermal keratinocyte viability on polymerized eShell 200 material was examined using the MTT assay.24
The MTT assay is based on reduction of a yellow tetrazolium salt (3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyl tetrazolium bromide) to a purple formazan dye by mitochondrial succinic dehydrogenase. The MTT assay uses mitochondria metabolism as a measure of cell viability. Cylindrical pellets of eShell 200 (n
= 3) were created by exposure of the polymer in PDMS molds (diameter = 6 mm; depth = 1 mm) using the same curing lamp and exposure time that were used to fabricate the microneedle arrays. Prior to conducting the MTT assay, the pellets were sterilized by ultraviolet B (UVB) radiation for 3 h, with both sides of the pellets receiving equal UVB light exposure. Cryopreserved human epidermal keratinocytes (Lonza, Walkersville, MD) were passed twice to initiate propagation, and 285,000 human epidermal keratinocytes were seeded in 10 ml of keratinocyte growth medium-2 (KGM-2), consisting of serum-free keratinocyte basal media, which is supplemented with bovine pituitary extract, epinephrine, GA-1000 (gentamicin-amphotericin), human epidermal growth factor, hydrocortisone, insulin, and transferrin (Lonza, Basel, Switzerland). The seeded human epidermal keratinocytes were then cultured at 37 °C and 5% CO2
until 80% confluency was obtained. The cells were then transferred to 96 well plates, in which pellets were placed at the bottom of the wells. The pellets and polystyrene culture wells were washed with 1 ml of KGM-2 prior to seeding. The pellets were held to the bottom of the culture wells by applying a drop of Akwa Tears®
(Akorn, Buffalo Grove, IL) to the bottom of the wells prior to pellet placement. The human epidermal keratinocytes were allowed to proliferate for 24 h before the MTT assay was performed. Human epidermal keratinocyte viability was determined by measuring absorbance at λ = 550 nm using a Multiskan RC ELISA plate reader (Labsystems, Helsinki, Finland). Cell viability on the eShell 200 polymer pellets was normalized to surface area and compared with growth on the surface of polystyrene wells with no pellets.
Human abdominal skin obtained from a surgical abdominoplasty was used to assess skin penetration of the microneedles. The use of this tissue received institutional approval according to the Declaration of Helsinki of the World Health Association. The skin was utilized within 24 h of removal. Subdermal fat was mechanically removed via dermatome to achieve uniform layers of full thickness skin. Stratum corneum and epidermal layers were obtained from full thickness skin by a heat separation method. The skin was immersed in distilled water at 60 ± 1 °C for 1 min. Then the stratum corneum and epidermis was gently peeled from the dermis using forceps. Isolated stratum corneum and epidermal layers were dried in a desiccator at ~30% relative humidity, wrapped in aluminium foil, and stored at −20 ± 1 °C until use. The integrity of the barrier was visually inspected to ensure that the stratum corneum and epidermal layers were unaltered during this process. This method of skin preparation has previously been used to examine microneedle-mediated drug delivery and other transdermal drug delivery techniques.25–28
Microneedles were inserted into the tissue using an electronic texture analyzer (Acquati, Arese, Italy). An eShell 200 microneedle array was placed on the stratum corneum and epidermis sample. The eShell 200 microneedle array was held in place using a polystyrene support and was compressed against the sample using a stainless steel probe (surface area = 1.13 cm2
). The probe was moved toward the sample at a rate of 300 mm/min until a maximum load of 4 N was obtained. The microneedles were held in place for 3 s and then removed. Similar protocols for examining microneedle insertion into human stratum corneum and epidermis have been reported in the literature.26,29–31
Optical microscopy was used to examine the microneedle–skin interaction.