Role of Constituents of Optison in Optison‐Mediated Gene Transfection Enhancement in Skeletal Muscle In Vivo

Objectives The mechanism by which Optison (an albumin‐shelled, octafluoropropane gas–filled microbubble contrast agent; Amersham Health, Amersham, England) enhances gene transfection in skeletal muscle in vivo with or without ultrasound (US) is unclear. The possible mechanisms were investigated by experimenting with different constituents, both with and without US. Methods Plasmid DNA (10 μg) encoding green fluorescent protein was mixed with Optison or its constituents dissolved in saline (in an equivalent concentration as in Optison) and injected into the tibialis anterior muscle of mice with or without adjunct US (1 MHz, 2 W/cm 2 , 30 seconds, and 20% duty cycle). The efficiencies of green fluorescent protein transgene expression were determined under different experimental conditions: (1) plasmid plus saline as a negative control; (2) plasmid plus Optison as a positive control; (3) plasmid plus heat‐treated Optison (without microbubbles); (4) plasmid plus human serum albumin; (5) plasmid plus N ‐acetyltryptophan; and (6) plasmid plus caprylic acid. Transfection efficiency was assessed by counting the maximum number of green fluorescent protein–positive fibers. Tissue damage was assessed by measuring the damaged area on serial sections. Results Heat‐treated Optison with or without US and albumin with US showed similarly high levels of transgene expression as Optison in mouse muscle without substantially increased tissue damage. N ‐Acetyltryptophan and caprylic acid had no effect on the delivery of plasmid green fluorescent protein into mouse muscle but instead showed the potential to increase tissue damage. Conclusions These data suggest that US and albumin separately potentiate transfection in this model. The combination of albumin and perfluoropropane is highly effective, which probably explains why Optison is so effective.