Focused Ultrasound-Mediated Delivery of Thiol-Decorated Gene Nanocarriers To Brain Tumors Yields Advantageous Tropism When Compared to CED

Introduction: Glioblastoma (GBM) is both highly aggressive and the most commonly diagnosed primary brain tumor in the central nervous system. Current treatment options include surgical resection, followed by chemotherapy and/or radiotherapy. However, efficient therapeutic delivery is hindered by the blood-brain (BBB) and blood-tumor (BTB) barriers. Thus, patient prognosis remains poor with a median survival of 15 months, highlighting the need for more targeted therapeutic delivery platforms. Nanoparticles (NPs) have shown some promising results in improving therapeutic delivery to solid tumors. To facilitate tumor cell-targeting, NPs are often modified with surface ligands designed to target molecules that are overexpressed by the target tissue. Exofacial thiols, or thiol (SH-) groups located on the cell membrane, are present on tumor cells due to dysregulated protein synthesis, increased redox activity, elevated pH, and high metabolic activity within the tumor microenvironment (TME). In this study, we developed a novel polymeric NP formulation with surface-bound thiol groups (SH-NP) that can exploit these exofacial thiols on tumor cells to improve targeting and transfection. Meanwhile, yet another strategy for targeting NPs to brain tumors is focused ultrasound (FUS)-mediated blood-tumor barrier (BTB) opening, a non-invasive modality capable that is well-known to augment NP delivery to brain tumors. Here, we deployed a new therapeutic strategy for GBM that combines SH-NPs with FUS-mediated BTB opening to further improve local therapeutic delivery.

Materials and

Methods: Thiol-decorated nanoparticles (SH-NPs) were characterized using dynamic light scattering. Free thiol groups on the surface of brain microvascular endothelial cells (bEnd.3) and cancer cells (GL261-Luc2) were evaluated using an Ellman’s colorimetric assay. 14 days after inoculation of C57BL6/J mice with GL261-Luc2 cells, mice were either treated with convection enhanced delivery (CED) of SH-NPs or FUS-mediated delivery of SH-NPs. For CED studies, mice were placed onto a stereotactic frame. A small incision was made on the head of the mouse to expose the skull. Using the existing burr hole as a guide, a Hamilton syringe was used to inject 20 µL of SH-NPs at an injection rate of 0.5 µL/min (20 µg of pDNA). For FUS-mediated BTB opening studies, we used a FUS Instruments skull landmark-guided RK-50 system coupled with intravenous delivery of SH-NPs (40 µg of pDNA). A clinically relevant FUS treatment regimen was employed using passive cavitation detection (PCD) monitoring and acoustic emissions feedback. To test whether the relative timing of SH-NP injection with respect to FUS affected transfection, SH-NPs were administered intravenously 15 minutes before (-15), at the time of (0), or 15 minutes after (+15) FUS. 2 days following treatment, tumor bearing brain quadrants were harvested and processed for flow cytometry to assess transfection efficacy in tumor cells (EphA2+), endothelial cells (CD31+), astrocytes (ASCA-2+), pericytes (CD146+), and leukocytes (CD45+). In both delivery regimens, major organs were harvested 2 days after SH-NP treatment and imaged for off-target transfection using the Spectral Instruments LagoX.

Results, Conclusions, and Discussions: Several characteristics of NPs facilitate their passage through the brain parenchyma, including small size, low positive charge, and dense PEGylation. We characterized SH-NPs, which are densely PEGylated, and found them to exhibit similar properties: a size of ~49 nm and a charge of 2 mV (Figure 1A-B). In-vitro, GL261-Luc2 cells exhibited a 10-fold increase in exofacial thiols when compared to non-cancerous bEnd.3 cells, suggesting the potential for targeting cancer cells through this mechanism (Figure 1C). FUS treatments were conducted in a three-point grid pattern to ensure complete tumor coverage (Figure 1D). Subsequent flow cytometric analysis, employing the gating strategy depicted in Figure 1E, aimed to elucidate and characterize the cellular tropism generated by transfection with SH-NPs. Decorating NPs with SH- groups did not impede transfection, as evidenced by successful transfection with both FUS and CED routes. Notably, FUS drove an ~10-fold increase in tumor cell transfection with SH-NPs when compared to IV-injected SH-NPs without FUS. FUS-mediated tumor cell transfection was comparable to that achieved with CED (Figure 1F). Notably, the efficacy of tumor cell transfection using SH-NPs remained consistent within the 30-minute intravenous injection time window, indicating that transfection efficiency is independent of SH-NP injection timing with respect to FUS (Figure 1G). Strikingly, although FUS and CED yielded similar SH-NP-mediated tumor cell transfection efficiencies, FUS markedly enhanced the transfection of other cell types within the TME when compared to CED (Figure 1H). Of particular note, FUS-mediated SH-NP delivery yielded exceptionally high transfection of endothelium and leukocytes, responses that could be leveraged therapeutically to improve immune cell infiltration into, and function within, the TME. Off-target organs exhibited minimal transfection when SH-NPs were administered IV, although some NP accumulation in lung was evident (Figure 1I). Our study underscores the potential of SH-NPs in targeted gene delivery for GBM and highlights the role of FUS in enhancing therapeutic efficacy and beneficially modulating tropism in the TME. These findings contribute insight into the optimization of non-invasive NP-based treatments for more effective and precisely targeted therapeutic strategies for GBM and other solid tumors.

Acknowledgements (Optional): Supported by an NSF GRFP to ACD and NIH R01CA279134, R01EB030409, and R01EB030744 to RJP.