Sonodynamic therapy using protoporphyrin IX encapsulated microbubbles inhibits tumor growth (2017)

Background: Sonodynamic therapy is a cancer treatment using drugs called “sonosensitizers” which, upon exposure to ultrasound (US), generate reactive oxygen species (ROS), thereby causing cell death. Despite the potential of sonodynamic therapy, many ideal sonosensitizers, such as protoporphyrin IX (PpIX), are hydrophobic compounds; poor water solubility limits systemic delivery and constrains clinical translation. We hypothesized that a PpIX-loaded lipid microbubble (MB) would have advantages over PpIX alone by ultimately allowing systemic delivery and also by locally concentrating US energy for greater PpIX activation. Accordingly, we developed an efficiently PpIX-loaded MB formulation and characterized its US-induced cytotoxicity in vitro and in an in vivo tumor model. Methods: PpIX was loaded in the hydrocarbon tail region of the lipid monolayer that forms the encapsulation shell of perfluorocarbon gas-filled MBs (“MBPpIX”). Suspensions of MBPpIX or free PpIX alone were exposed to various US conditions (1 MHz, 1-8 W/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> spatial average pulse average intensity (I <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">SAPA</sub> ), 1-10% duty cycle), and ROS generation was measured with Amplex Red. MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> or equivalent amount of free PpIX (dissolved in DMSO, final PpIX concentration: 2.9 μM) was added to cultured squamous cell carcinoma (SCC-VII) cells, followed by US treatment for 2 min and alamarBlue Assay for viability 24 hrs post US. To evaluate anti-tumor effect in vivo, SCC-VII tumor-bearing mice received intratumoral injection of 1×10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">8</sup> MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> , equivalent dose (18.4 μg) of PpIX only, or blank MB (“MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Blank</sub> ”), followed by 4 min of US treatment (1 MHz, I <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">SAPA</sub> =3 W/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> , 10% duty cycle). Tumor volume was serially imaged and quantified by high-resolution 3D US. Results: A stable MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> formulation was achieved with high drug loading (mean MB diameter 1.6±0.1 μm; 184±49 μg PpIX per 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">9</sup> MB). ROS was generated with MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> and free PpIX, in an US-dependent manner. For both MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> and free PpIX, US-dependent cytotoxicity was observed; e.g. for MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> , more than 90% of SCC-VII cells were killed with US at I <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">SAPA</sub> =2 or 4 W/cm <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">2</sup> and 10% duty cycle. In vivo, MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> + US caused the greatest tumor growth inhibition, with doubling time (2.7±0.5 days) more prolonged compared to that for US-treated tumors injected with only PpIX (2.1±0.3 days; p=0.02) or MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">Blank</sub> (1.7±0.5 days; p=0.01). Conclusions: These data indicate that in conjunction with pulsed US, MB <sub xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">PpIX</sub> is an effective sonodynamic therapy platform that may be superior to PpIX alone. Our findings set the stage for non-invasive sonodynamic therapy using systemic delivery of PpIX, whereby PpIX-carrying lipid MBs allow circulation of this otherwise insoluble compound, while also focusing and augmenting US energy for enhanced sonodynamic effect.