RNA-based therapies offer unique advantages for treating brain tumors. However, tumor penetrance and uptake are hampered by RNA therapeutic size, charge, and need to be “packaged” in large carriers to improve bioavailability. Here, we have examined delivery of siRNA, packaged in 50-nm cationic lipid-polymer hybrid nanoparticles (LPHs:siRNA), combined with microbubble-enhanced focused ultrasound (MB-FUS) in pediatric and adult preclinical brain tumor models. Using single-cell image analysis, we show that MB-FUS in combination with LPHs:siRNA leads to more than 10-fold improvement in siRNA delivery into brain tumor microenvironments of the two models. MB-FUS delivery of Smoothened (SMO) targeting siRNAs reduces SMO protein production and markedly increases tumor cell death in the SMO-activated medulloblastoma model. Moreover, our analysis reveals that MB-FUS and nanoparticle properties can be optimized to maximize delivery in the brain tumor microenvironment, thereby serving as a platform for developing next-generation tunable delivery systems for RNA-based therapy in brain tumors.

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Despite the challenges in treating glioblastomas (GBMs) with immune adjuvants, increasing evidence suggests that targeting the immune cells within the tumor microenvironment (TME) can lead to improved responses. Here, we present a closed-loop controlled, microbubble-enhanced focused ultrasound (MB-FUS) system and test its abilities to safely and effectively treat GBMs using immune checkpoint blockade. The proposed system can fine-tune the exposure settings to promote MB acoustic emission–dependent expression of the proinflammatory marker ICAM-1 and delivery of anti-PD1 in a mouse model of GBM. In addition to enhanced interaction of proinflammatory macrophages within the PD1-expressing TME and significant improvement in survival ( P  < 0.05), the combined treatment induced long-lived memory T cell formation within the brain that supported tumor rejection in rechallenge experiments. Collectively, our findings demonstrate the ability of MB-FUS to augment the therapeutic impact of immune checkpoint blockade in GBMs and reinforce the notion of spatially tumor-targeted (loco-regional) brain cancer immunotherapy.

RNA-based therapies offer unique advantages for treating pediatric brain tumors. However, the systemic delivery remains a major problem due to degradation of unmodified RNA in biological fluids, poor brain accumulation, and poor cancer cell uptake or escape from the endosomal lipid bilayer barrier. While nanoparticle encapsulation can prolong circulation time and facilitate cellular uptake, their accumulation in brain tumor remains particularly poor due to their low permeability across the blood-brain barrier and limited intratumoral penetration. Focused ultrasound, when combined with circulating microbubbles (MB-FUS) provides a physical method to transiently modulate the brain tumor microenvironment (TME) and improve nanoparticle delivery. Here, we have examined the delivery of siRNA targeting the Smoothened (SMO) pathway, packaged in 50 nm cationic lipid-polymer hybrid nanoparticles (cLPH:siRNA-SMO), combined with MB-FUS in murine SmoA2 sonic hedgehog (SHH) medulloblastoma. At 30 hours after treatment, we observed the depletion of the SMO protein target, responsible for driving SHH medulloblastoma formation and growth, in mice that had received treatment with MB-FUS and cLPH:siRNA-SMO, but not with cLPH:siRNA-SMO alone. We also confirmed that SMO protein depletion was spatially achieved in the tumor regions with detected cLPH:siRNA-SMO using FISH assay, and that there was 15 fold induction of tumor cell apoptosis compared to tumors in mice that had received cLPH:siRNA-SMO alone. The limited induction of apoptosis was observed with either cLPH:siRNA (non-targeting) or MB-FUS and cLPH:siRNA (non-targeting), suggest that the observed apoptosis induction in the SmoA2 model was the direct result of SMO depletion rather than nonspecific effects of MB-FUS or cLPH:siRNA. Our findings provide a paradigm shift in drug delivery in brain tumors, where physical methods and nanotechnology are tuned together to develop rational strategies for the effective delivery of nucleic acids in brain tumors.

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