In vitro Skin Penetration Studies

Skin from freshly excised porcine ears was obtained from a local slaughterhouse (Fribordog, Bariri, SP, Brazil), carefully dissected to remove the subcutaneous fat and adhering tissues, washed under running tap water, stored at – 80 °C and used within one month.

Before the experiments, the skin was dermatomized at 700-μm of thickness and tightly fitted in a Franz-type diffusion cell (diffusion area of 0.9 cm²), with the dermis and stratum corneum facing the receptor and donor compartment, respectively.

Stratum corneum integrity was determined by placing Ag/AgCl electrodes (In Vivo Metrics, Healdsburg, CA, USA) in contact with PBS solutions that bathed the donor and receptor compartments separated by the skin mounted on the diffusion cell and subjecting this circuit to an alternating current (100 mV, 10 Hz) using a 20 MHz function/arbitrary waveform generator (Agilent, 33220A, Santa Clara, USA). The electric current capable of passing through the skin was measured using a multimeter (Minipa, ET 1450, São Paulo, SP, Brazil) and used to calculate the skin resistance based on the Ohm’s law. Resistivity was obtained by multiplying the resistance by the area of skin available for permeation. Only the skin samples that showed a resistivity of 35 > kΩcm² were used in this study.³⁷

The receptor compartment (16 mL) was filled with PBS isotonic buffer solution (10 mmol.L⁻¹, pH 7.4, 0.9% NaCl) containing 1% (w/v) SLS to guarantee sink conditions (ZnPc solubility in this medium is 12.5 ± 0.1 μg.mL⁻¹),²⁵ kept at room temperature and under magnetic stirring. The influence of the receptor solution on the integrity of the stratum corneum was verified by subjecting the system to the passage of alternating electric current and monitoring resistivity for up to 24 h.

The optimized ZnPc-loaded micelles used in the skin penetration experiments were prepared from 20 mg of DSPE-PEG and 210 μg of ZnPc dissolved in 3 mL chloroform to form the film in the wall of the round bottom flask after organic solvent evaporation, which was hydrated with 3 mL of Tween 80: Span 80 (1:3 mol) at 0.5% in HEPES pH 7.4. ZnPc-loaded micelles containing 15 ± 3 μg mL⁻¹ ZnPc were used in the experiments.

The penetration of ZnPc into the skin was evaluated from 3 different series of experiments. In the first one, 1 mL of ZnPc-loaded micelles was applied to the donor compartment for 6 h and 24 h to evaluate passive ZnPc penetration. In experiments performed for 24 h, however, SLS was not added to the receptor solution to avoid altering the integrity of the skin (Supplementary material, Figure S1.1). In the second series, the skin was pretreated with 20 kHz LFU (Sonics & Materials, VCX 500, Newtown, CT USA) at I_sata of 10 ± 0.5 W/cm², duty cycle 50% (5 s on, 5 s off), tip displacement from the skin surface of 10 mm immersing in 1 mL of 1% HEC hydrogel as the coupling medium. The skin was pretreated until it reached a resistivity of 1.0 ± 0.5 KΩ.cm2.³⁷ To minimize thermal effects, the coupling medium was replaced after each minute of treatment.³⁷ After the pretreatment, 1 mL of ZnPc-loaded micelles was applied to the donor compartment for 6 h to evaluate ZnPc penetration under the influence of LFU pretreatment. Finally, the third series of experiments was performed using ZnPc-loaded micelles as the coupling medium. The parameters and application of the LFU were the same as those previously used in the pretreatment protocol. After skin attained 1.0 ± 0.5 KΩ.cm², 1 mL of ZnPc-loaded micelles was applied to the donor compartment for 6 h to evaluate ZnPc penetration under the influence of the LFU-ZnPc simultaneous protocol.

After 6 or 24-hour contact of the ZnPc-loaded micelles with the skin, the skin surfaces were carefully rinsed with distilled water to remove the excess formulation and carefully wiped with tissue paper. The stratum corneum was separated from the skin using a validated tape stripping technique with 15 pieces of adhesive tape (Scotch shipping packaging tape 3M, St. Paul, MN, USA).²⁵ The tape strips containing the stratum corneum were all together immersed in 5 mL of DMSO, vortex-stirred for 2 min, and sonicated for 30 min in an ultrasonic bath. DMSO phase was filtered through a 0.45 μm membrane filter of PTFE, and the resulting filtrate was analyzed by spectrofluorimetry to determine the concentration of ZnPc in the stratum corneum.²⁵ The remaining skin was placed in a zip-locked plastic bag and immersed in hot water at 60°C for 1 min to separate, with the help of a spatula, the epidermis without stratum corneum (viable epidermis) of the dermis. Fragmented epidermis and dermis were separately vortex-mixed (IKA Works, Wilmington, NC, USA) for 2 min in 2 mL of DMSO, homogenized using a tissue homogenizer (IKA Works, T10 basic, Wilmington, NC, USA) for 1 min at 13,500 rpm and bath-sonicated (Quimis, Q335 model, 40 kHz, São Paulo, Brazil) for 30 min. The resulting dispersion was centrifuged at 20,000x g for 10 min, and the supernatant was filtered through a 0.45 μm PTFE membrane. Finally, the ZnPc was assayed by spectrofluorimetry to determine its penetration in the viable epidermis and in dermis according to Equation 1.

To compare passive and sonophoretic penetrations, the ZnPc flux through the skin was determined at the steady-state (after 24 h of passive experiment and 6 h of experiments with LFU) by dividing the amount of drug in the dermis by the time of penetration.

The contribution of convection and acoustic transmission in the penetration of ZnPc by the LFU-ZnPc simultaneous treatment was estimated by the difference between the ZnPc flux after LFU simultaneous treatment and the flux after LFU pretreatment.

For confocal fluorescence microscopy, in vitro 6-h passive and sonophoretic penetrations with ZnPc micelles were carried out as described before. Also, the skin was also subjected to LFU-ZnPc simultaneous treatment without 6-h subsequent exposure to the micelles containing ZnPc. After treatments, the skin surfaces were carefully rinsed with distilled water to remove any excess formulation. The skin penetration areas were excised, soaked in Tissue-Tek^® (O.C.T. Compound) inside plastic molds, frozen in acetone and dry ice, and subsequently stored at – 80°C. Cryosections of 15-μm of thickness, perpendicular to the skin surface were made using a cryostat (Leica CM1860, Illinois, EUA). All slices received Fluoromount to prevent photobleaching during analysis. A Leica TCS SP8 confocal microscope (Mannheim, Germany) with a 20x immersion objective was used for confocal fluorescence microscopy. Samples were excited with a laser at 638 nm and the fluorescence was monitored at 640–800 nm. Untreated skin was used to adjust the parameters of the microscope so that the autofluorescence of the tissue did not interfere in the analyses.