Mechanism of ultrasound enhanced porphyrin cytotoxicity: free radical and hematoporphyrin effects

Intense ultrasound beams have the potential for treating malignant tumours when combined with sonodynamic sensitizers. Some of these agents, e.g. porphyrins, are also used for photodynamic therapy. However, the experimental evidence is inconsistent. This work attempts to discover if the yield of free-radicals such as .OH and .H which are produced by transient cavitation could explain the killing of Chinese hamster ovary (CHO) cells in vitro with and without sonodynamic agents. CHO cells were irradiated with ultrasound beams in phosphate buffered saline [PBS] or in growth medium, and the immediate cell lysis and loss of cell colony forming ability measured. Under the authors' specific conditions, in which the standing wave patterns were minimized, a general correlation was observed between the transient cavitation, free-radical production, and cytotoxicity. However, the yield of free-radicals was much too small to explain the cell killing observed. The authors conclude that cytotoxicity is not linked to attack from free-radicals formed outside the cells. In their experiments, immediate cell lysis is closely linked to the transient cavitation which is known to produce shear forces which disrupt cellular membranes. The authors hypothesize that the loss of cell colony forming ability is also linked to damage of cellular membranes. In further experiments, CHO cells were irradiated with ultrasound beams in different media with and without hematoporphyrin (Hp) to ask the question: is there enough toxic singlet oxygen, formed from energy transfer from the excited Hp to oxygen, to explain the cytotoxicity? The addition of Hp to CHO cell insonations did not change the amount of immediate lysis or cytotoxicity as measured by colony growth. During Hp experiments under conditions which mimic a real PDT treatment, singlet oxygen fluorescence was produced by laser excitation but none was produced by the ultrasound beam. The authors conclude that the cytotoxicity is not linked to free-radical attack, or singlet oxygen, but hypothesize that the cytotoxicity is due to the transient cavitation producing shear forces which disrupt cellular membranes.