Influence of the low-frequency ultrasound on the skin penetration of zinc phthalocyanine formulations and its potential for the sonodynamic therapy of skin tumors

Author(s)

Yugo Araújo Martins

• Document type: Master's Dissertation
• Press: Ribeirão Preto.
• Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
• Defense date: 2019-09-10

Examining board members

Renata Fonseca Vianna Lopez; Rodrigo da Cunha Bisaggio; Théo Zeferino Pavan

• Advisor: Renata Fonseca Vianna Lopez

Abstract

Introduction: Sonodynamic therapy (TSD) is a new therapeutic modality for the noninvasive treatment of skin cancer based on the association of ultrasound and sonosensitizing agents. TSD results in cell death through acoustic cavitation-dependent mechanisms, which are not yet fully understood. This study aimed to evaluate the influence of the low-frequency ultrasound (LFU) on the skin penetration of a model sonosensitizing agent, zinc phtalocyanine (ZnF) and on the generation of oxygen reactive species (ROS) and free radicals, which are important for the TSD efficacy. The characteristics of the ZnF carrier systems in the LFU-mediated penetration were also investigated. Methods: Micelles of ZnF based on polyethylene glycol 2000-conjugated 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE-PEG) were obtained after determining the critical micellar concentration (CMC) of DSPE-PEG by the method of pyrene. Micelles of ZnF were prepared by hydration method of the lipid film, optimized by the Box-Behnken experimental design and physicochemical and morphologically characterized. The cavitation activity of the coupling media used in the in vitro penetration experiments was estimated using the KI dosimeter method. Studies of passive penetration in porcine skin under the influence of LFU in the pre-treatment and simultaneous protocols were carried out, and the distribution of ZnF in the different layers of the skin was quantitatively monitored and qualitatively demonstrated by confocal microscopy. In the pre-treatment, hydrogel of hydroxyethylcellulose (HEC) was used as coupling medium. After pre-treatment and simultaneous protocols, the micelles and the emulsion containing ZnF were put in contact with the LFU-treated skin in order to investigate the effect of the characteristics of the formulations on the drug penetration. The LFU parameters used in the experiments were 20 kHz, 10 W/cm2 in the pulsatile mode set in 5 s on and 5 s off. The skin was irradiated until the resistivity of the stratum corneum reached 1 k?.cm2.The generation of hydroxyl radicals, singlet oxygen and lipid peroxidation by the LFU in the presence of ZnF was also evaluated. Results: The CMC value of the DSPE-PEG in 20-mmol/L HEPES buffer solution (pH 7,4) was 2,0 x 10-5 mol/L. The micelles of ZnF showed particle size, polydispersion index (PdI), zeta potential and ZnF concentration of 138?10 nm, 0,25?0,01, -27?1 mV e 13?2 ?g/mL, respectively, and spherical shape. The hydrogel of HEC and the blank micelles used as coupling media increased the cavitation activity by 2.6 and 1.8-fold, respectively, in comparison to the aqueous solution. The amount of ZnF quantified in the dermis after 6h-passive permeation was approximately 4-fold higher when delivery by the emulsion in comparison to the micelles. However, in the pre-treatment and simultaneous protocols with LFU, micelles increased in 21 and 7-fold the penetration of ZnF compared to the emulsion. The simultaneous treatment of the skin with the micelle of ZnF yielded the highest amount and more homogeneous distribution of the drug in all skin layers. The LFU irradiation increased the oxidation of iodide ions by the ultrasound-generated hydroxyl radicals in 26-fold and significantly generated singlet oxygen compared to the respective control groups. Moreover, LFU irradiation in the skin, previously underwent to 6h-passive permeation with the micelles of ZnF, duplicated the lipid peroxidation. Conclusion: The potential of the LFU associated with the micelles of ZnF to promote the skin penetration of the drug and generate ROS and free radicals for the sonodynamic therapy of skin tumors has been demonstrated (AU)

• FAPESP's process: 17/17442-7 - Evaluation of the low-frequency ultrasound potential for the sonodynamic therapy of skin cancer
• Grantee: Yugo Araújo Martins
• Support Opportunities: Scholarships in Brazil - Master