Sonodynamic therapy using protoporphyrin IX encapsulated microbubbles inhibits tumor growth

Abstract

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/cm2 spatial average pulse average intensity (I SAPA ), 1–10% duty cycle), and ROS generation was measured with Amplex Red. MB PpIX 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 1×108 MB PpIX , equivalent dose (18.4 μg) of PpIX only, or blank MB (“MB Blank ”), followed by 4 min of US treatment (1 MHz, I SAPA =3 W/cm2, 10% duty cycle). Tumor volume was serially imaged and quantified by high-resolution 3D US. Results: A stable MB PpIX formulation was achieved with high drug loading (mean MB diameter 1.6±0.1 μm; 184±49 μg PpIX per 109 MB). ROS was generated with MB PpIX and free PpIX, in an US-dependent manner. For both MB PpIX and free PpIX, US-dependent cytotoxicity was observed; e.g. for MB PpIX , more than 90% of SCC-VII cells were killed with US at I SAPA =2 or 4 W/cm2 and 10% duty cycle. In vivo, MB PpIX + 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 Blank (1.7±0.5 days; p=0.01). Conclusions: These data indicate that in conjunction with pulsed US, MB PpIX 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.