Caffeine powder

IBMX, forskolin, phenylthiourea (PTU), sodium hydroxide (NaOH), DMSO, L-3,4-dihydroxyphenylalanine (L-DOPA), caffeine powder, staurosporine, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and CellLytic™ lysis buffer were purchased from Sigma (St Louis, MO, USA). Isofraxidin was purchased from ChemFaces Biochemical (Hubei, China).

Tough 1500 photopolymer resin material was used to 3D-print the MN arrays in this research. This material supports printing resolutions of 50 and 100 microns in the layer thickness [65 (link)]. Moreover, this material is resilient, biocompatible, tough, and durable. In addition, Tough 1500 Resin is certified to be safe for skin contact, which makes it an ideal material for fabricating biomedical devices. The anhydrous ethanol obtained from Fisher Chemical was used to cure the 3D-printed MN array. Dow Sylgard™ 184 Silicone Elastomer, a polydimethylsiloxane (PDMS), was obtained from Ellsworth Adhesives, Germantown, WI, USA. PDMS material was used to develop the female MN array mold. Gantrez™ AN-169, alternating copolymers of methyl vinyl ether (MVE) and maleic anhydride, was sourced from Ashland. Poly (ethylene glycol) with average Mn20,000 and Caffeine powder were obtained from Sigma Aldrich, Burlington, MA, USA. The combination of Gantrez™ and Caffeine powder (X w/v %) was used to fabricate the biodegradable MN array patch. Parafilm M^®. (Sigma Aldrich), a flexible thermoplastic sheet with 0.13 mm thickness, was used as a skin simulator (artificial skin) for insertion studies.

The following chemicals were used: ketamine hydrochloride (Laboratório Köing, Santana de Parnaíba, SP, Brazil), xylazine hydrochloride (Laboratório Vallée, Montes Claros, MG, Brazil), lidocaine (Laboratório Hipolabor, Sabará, MG, Brazil) and caffeine powder (Sigma), in the form of crystals diluted and 0.9% saline solution.

Human GBM cell line U87-MG (American Type Tissue Collection, Manassas, VA, USA) was cultured in minimum essential medium-alpha (MEM-α) supplemented with 10% heat-inactivated foetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin (all from Gibco, Life Technologies, Inc., Carlsbad, CA, USA) in a humidified incubator with 95% air atmosphere and 5% carbon dioxide at 37°C. Caffeine powder was obtained from Sigma, and stock solution was prepared in culture medium at the concentration of 32 mM and kept in 4°C. TMZ was obtained from Schering-Plough (Kenilworth, NJ, USA) and dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Saint Louis, MO, USA) at the concentration 100 mM, further diluted in culture medium to 10 mM, and stored in -20°C. For treatment, caffeine and TMZ were further diluted in medium to the final concentrations as stated below.
A four-day treatment was divided into two phases: one-day pretreatment and a subsequent three-day treatment of either TMZ alone, caffeine alone, or both (Figure 1). Overall, six arms of cells were set up accordingly. The dosage of caffeine was 1 mM as determined by MTT assay, and the dosage of TMZ we opted was its IC₅₀ value defined in U87-MG cells in our previous work [2 (link)].