Study of sonodynamic therapy for the treatment of cutaneous melanoma in an animal model

• Ano de defesa: 2025
• Autor(a) principal: Ayala, Erika Toneth Ponce
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Biblioteca Digitais de Teses e Dissertações da USP
• Palavras-chave em Português: 5-aminolevulinic acid; Ácido 5-aminolevulínico; Cutaneous melanoma; Melanoma cutâneo; Photodynamic therapy; Protoporfirina IX; Protoporphyrin IX; Sonodynamic therapy; Terapia fotodinâmica; Terapia sonodinâmica
• Link de acesso: https://www.teses.usp.br/teses/disponiveis/76/76134/tde-05052025-081918/

Cutaneous melanoma is one of the most aggressive and lethal forms of skin cancer. Although surgical excision remains the standard treatment, it can be suboptimal and invasive in some instances. As a result, alternative therapeutic strategies are being explored, including dynamic therapies such as sonodynamic therapy (SDT) and sonophotodynamic therapy (SPDT). SDT relies on the interaction between a sonoactive molecule, known as a sonosensitizer (SS), and low-intensity ultrasound in an oxygenated environment. SPDT, on the other hand, combines photodynamic therapy (PDT) with SDT to enhance therapeutic efficacy through synergistic effects. Both SDT and SPDT have shown potential as non-invasive cancer treatments, as demonstrated in various preclinical studies, where sonomechanical and sonochemical effects, such as damage to tumor cell membranes and the production of reactive oxygen species (ROS), play key roles. However, the potential of SDT and SPDT for treating cutaneous melanoma has yet to be explored. In this work, two in vitro studies were initially conducted, followed by two in vivo studies. The in vitro studies aimed to investigate the interaction between low-intensity ultrasound and several well-established organic sensitizers widely reported in the literature, such as protoporphyrin IX (PpIX), chlorin e6 (Ce6), methylene Blue (MB), and curcumin. For this purpose, the generation of two types of reactive oxygen species (ROS) was evaluated during photo-, sono-, and sonophotodynamic actions, and the degradation products resulting from photo- and sonodynamic actions were compared. The combined action induced only a slight increase in the quantum yield of singlet oxygen (1O2) from photodynamic therapy. Therefore, we conclude that, under the experimental conditions and methodology used in this study, 1O2 generation is unlikely to be the predominant mechanism underlying sonophotodynamic therapy (SPDT), in contrast to photodynamic therapy (PDT). On the other hand, hydroxyl radical (·OH) generation induced by photodynamic action was significantly enhanced by the combined treatment, suggesting that ·OH production is a key mechanism during SPDT. Finally, PpIX and Ce6 proved to be more effective sonophotosensitizers compared to the other molecules evaluated. The in vivo studies aimed to evaluate the effects of a dynamic and clinically viable therapeutic protocol for treating a pigmented murine intradermal melanoma model using a free organic sensitizing molecule. Based on the in vitro results, the precursor of PpIX, 5-aminolevulinic acid (ALA), was selected. Initially, the most efficient route of ALA administration was assessed to maximize PpIX deposition in cutaneous melanoma tumors in mice. Finally, SPDT was evaluated in this model through three distinct sub-studies: (1) application of SPDT in tumors at two different stages, stage 1 (diameter: 11.5 mm) and stage 2 (diameter: 22.5 mm); (2) investigation of potential synergistic effects of combining SDT and PDT; and (3) comparison of two ultrasound delivery methods, one using coupling gel and the other employing a conical aluminum waveguide. Based on the results obtained, the most effective ALA administration routes were intratumoral injection with a 2-hour incubation period and intraperitoneal injection with a 3-hour incubation period. The latter was chosen for therapeutic testing in mice due to the better sensitizer distribution in the tumor region. Sub-study 1 demonstrated that ALA-mediated SPDT has therapeutic potential for treating tumors at both stages. In sub-study 2, no significant synergistic effects were observed from the combination of SDT and PDT, indicating that, under the conditions employed, SPDT alone was more effective for treating cutaneous melanoma. Finally, Sub-study 3 revealed that for stage 2 tumors, both the waveguide and direct ultrasound application showed similar efficacy. However, for stage 1 tumors, the waveguide proved advantageous by focusing the ultrasound waves on small tumor regions while minimizing damage to the surrounding healthy tissue.