Preparation of transfersomes loaded by eosin

Transfersomes loaded by EY were prepared as previously described in our previous work [14]. Briefly, the lipid (lecithin from soybean oil) and the edge activator (sodium deoxycholate) were dissolved in a mixture of chloroform: methanol 2:1. The organic solvents were then evaporated under vacuum in a rotary evaporator (Heidolph-Elektro GmbH + Co KG, Germany) rotating at 90 rpm at 45 °C. The rotation is continued till complete evaporation of the solvents and the formation of a uniform thin lipid film. The obtained lipid film was then hydrated by phosphate-buffered EY solution (PBS-EY) and left in the rotary evaporator for a further 1 h. The lipid: edge activator: EY ratio was set as 10:1:1. The transfersomes dispersion was sonicated for 5 min in a water bath sonicator and was stored in the refrigerator for further use.

Characterization of the prepared transfersomesI. Morphology and shape

The diluted transfersomal dispersion was examined by a fluorescence microscope (Olympus BX51), supplied by a 75-W xenon lamp as an excitation source.

Moreover, the shape of the prepared transfersomes was examined under transmission electron microscopy (TEM, Jeol, Ltd., Tokyo, Japan) after negative staining.

II. UV–visible spectroscopy

The absorption spectrum of (PBS-EY) solution and EY-transfersomes dispersion were recorded by double beam spectrophotometer (RayLeigh UV-2601) at wavelength range 400–700 nm, using PBS solution as a blank reference.

III. Encapsulation efficiency (EE)

The unloaded EY was separated from the loaded transfersomes by centrifugation at 10,000 rpm at 5–6 °C (Centrikon T-42 K, Kontron Instruments, UK). The precipitated loaded transfersomes were dissolved in ethanol, to extract EY from them, and diluted by PBS (pH 7.4). The concentration of the extracted EY was measured spectrophotometrically at 520 nm (Rayleigh UV-2601) from a previously established standard calibration curve in PB-ethanol solution. Finally, the EE was determined as the following: EE % = (the calculated EY concentration extracted from the transfersomes/initial EY concentration used in preparation) X100.

IV. Particle size and zeta potential

The mean particle size and zeta potential of the prepared transfersomes were measured by the Zetasizer system (Malvern Instruments Ltd., Malvern, UK) after ten folds dilution with double distilled water at laser wavelength 632 nm, a temperature of 23 °C, scattering angle of 14.1°, and a refractive index of 1.33.

V. In-vitro release of EY from the prepared transfersomes

Three aliquots (n = 3) of the transfersomes suspension were dispersed in PBS (pH 7.4) and incubated separately at 37 °C under continuous stirring in a water bath. Each of the incubated dispersions was centrifuged for 10 min at 10,000 rpm at preset intervals. The precipitates were re-dispersed in fresh PBS and the concentration of released EY in the supernatants was measured by measuring the absorbance at 520 nm. Finally, the cumulative EY released percentage was determined as a function of time.

Preparation of transfersomal eosin hydrogel

The prepared EY-loaded transfersomes were incorporated into 5% Na-CMC hydrogel. A volume of transfersomal suspension, corresponding to 20 mg EY, was completed to 100 ml distilled water. Then, 5 gm of Na-CMC was added portion-wise with continuous stirring till full dispersion. Methylparaben (0.1%) and propylparaben (0.2%) were then added with continuous stirring to serve as preservatives. The gel was left for 24 h for complete swelling and to ensure the absence of any aggregates or air bubbles.

Clinical casesPatients

The study was conducted at the dermatology clinic at the National Institute of Laser Enhanced Sciences (NILES), Cairo University, Egypt, after the approval of the board of the research ethics committee of the Institute (approval no: CU-NILES/24/21). The study protocol conformed to the guidelines of the Declaration of Helsinki. This case study was conducted on 6 patients, representing 6 case reports. The demographic data of the patients are listed in Table 1. A written informed consent was obtained from every patient. The authors affirm that one of the participants provided informed consent for publication of the images in Fig. 4a and b.

Table 1 Patients characteristic

Exclusion criteria included children, pregnant or lactating females.

Treatment protocols

The palms were subjected to 2 passes of fractional CO2 laser (Bison, Italy) with the following parameters; scanning area 3 ~ 20 mm, Y: 3 ~ 20 mm, the fluence 7.5 mJ/cm2, pulse duration 500 µs and dot density 0.8. Afterward, the transfersomal EY gel was applied for 5 min.

The affected sites were then irradiated using an intense pulsed light (IPL, EPI-C PLUS; Espansione Group, Bologna, Italy), as a light source to activate the photosensitizer, fitted with a 550 nm filter that matches the maximum absorption peak of EY (2.5 × 4.5 cm spot area, total area 11.25 cm2, 20 ms pulse duration, and 25 J/cm2 fluence). Sessions were performed once per week, for 6 weeks. The assessment was done by starch iodine test to determine the area of hyperhidrosis before and after treatment. The sweating intensity was determined based on the color intensity of the starch iodine test according to the 5-grades Sweating Intensity Visual Scale [11], where Grade 0 indicates no sweating, grade 1 indicates minimal sweating, grade 2 indicates mild sweating, grade 3 indicates moderate sweating, grade 4indicates intense sweating, and grade 5 indicates over-sweating.

Moreover, patient satisfaction was measured by a scoring system: 0 (unsatisfied), 1 (partially satisfied), and 2 (very satisfied).