Abstract
Medulloblastoma is the most common malignant paediatric brain tumour, with ~30% mediated by Sonic hedgehog signalling. Vismodegib-mediated inhibition of the Sonic hedgehog effector Smoothened inhibits tumour growth but causes growth plate fusion at effective doses. Here, we report a nanotherapeutic approach targeting endothelial tumour vasculature to enhance blood–brain barrier crossing. We use fucoidan-based nanocarriers targeting endothelial P-selectin to induce caveolin-1-dependent transcytosis and thus nanocarrier transport into the brain tumour microenvironment in a selective and active manner, the efficiency of which is increased by radiation treatment. In a Sonic hedgehog medulloblastoma animal model, fucoidan-based nanoparticles encapsulating vismodegib exhibit a striking efficacy and marked reduced bone toxicity and drug exposure to healthy brain tissue. Overall, these findings demonstrate a potent strategy for targeted intracranial pharmacodelivery that overcomes the restrictive blood–brain barrier to achieve enhanced tumour-selective penetration and has therapeutic implications for diseases within the central nervous system.
Main
Site-directed drug delivery to brain parenchymal tissue is a desirable but elusive goal due to the distinct and highly regulated blood–brain barrier (BBB). The BBB is comprised of a network of specialized endothelial cells, pericytes and astrocytes that prevent material extravasation1. Characteristic tight junctions between adjacent brain endothelial cells block paracellular transport, and the passive entry of molecules is constrained to a narrow window of size and lipophilicity. These restrictive physical and functional barriers inhibit drug exposure to intracranial tissues2. Hydrophilic small-molecule drugs are routinely excluded from the brain by tight junctions and, while many lipophilic drugs are capable of entry via passive diffusion, penetration into diseased brain tissue is inefficient and typically requires high drug doses, often resulting in dose-limiting systemic toxicity3. As such, the integrity of the BBB can considerably impact treatment efficacy. This is well evidenced in medulloblastoma wherein patients in the Sonic hedgehog medulloblastoma subgroup (SHH-MB) suffer worse clinical outcomes due, in part, to an intact BBB limiting the entry of drugs into the brain at therapeutic concentrations4,5,6. In addition, targeted inhibition of the SHH effector Smoothened (SMO) via vismodegib causes premature bone growth plate fusion in paediatric patients, probably as a result of the high doses required for therapeutic efficacy7,8.
Given the many limitations for the passage of small molecules across the BBB, nanoparticles have been explored as a vehicle to improve delivery into brain tissues9. To date, much of this work has focused on strategies that enhance passive mechanisms of transport for drug-loaded nanoparticles across the BBB. For instance, in diseases that result in a compromised BBB such as glioblastoma, nanostructures have been observed to extravasate through the leaky vasculature to accumulate at tumour sites10. Also, researchers have implemented analogous approaches to improve drug delivery past an intact BBB by developing strategies that first disrupt this barrier11,12,13. However, by permitting unregulated passage across the BBB, such approaches not only abrogate the homeostatic functions of the BBB but potentially expose the brain to harmful toxins and pathogens. Alternative approaches for diseases such as SHH subgroup medulloblastoma that retain BBB integrity have utilized nontargeting nanocarriers to extend systemic circulation of small-molecule drugs, with only partial improvement of on-target toxicity profiles at high doses14. Importantly, recent work has suggested that passive entry of nanoparticles into solid tumours through gaps between endothelial cells represents a minor mechanism of entry and that up to 97% of transport is through an active process through endothelial cells15. However, the molecular mechanism of this active transcellular transport across endothelial barriers has not yet been elucidated and little is known of whether this transendothelial nanoparticle transport occurs at the BBB.
In this study, we investigated active transcellular transport mechanisms to enhance drug delivery across an intact BBB specifically to brain tumour tissue. We and others previously demonstrated targeting of fucoidan-based nanoparticles to P-selectin on activated endothelial cells and found that P-selectin expression on endothelial cells may be enhanced by radiotherapy (RT) to effect greater nanoparticle accumulation in tumour sites16,17. Herein, we found that P-selectin facilitates material transendothelial transport across an intact BBB via caveolin-1 (Cav1)-mediated transcytosis. Using a genetic mouse model of SHH-MB with an intact BBB, we found that P-selectin targeting results in active transport in tumour endothelium to enable delivery of fucoidan-based nanoparticles selectively into the tumour microenvironment, which is enhanced by RT. Fucoidan nanoparticles encapsulating the Smoothened inhibitor vismodegib (FiVis) exhibited potent effector inhibition at low drug doses, striking antitumour efficacy and attenuated on-target bone-related toxicities. These findings demonstrate a targeted approach for improving the therapeutic index of vismodegib for SHH-MB and present a potent adjuvant strategy for delivery of drugs to treat brain tumours in combination with standard RT. Furthermore, we report an active mechanism of transendothelial transport that can be exploited to improve drug delivery across activated brain endothelial cells at sites of intracranial disease in conditions with an intact BBB.
Low-dose irradiation enhances P-selectin on tumour vasculature
We first characterized the brain vasculature in a genetically engineered mouse (GEM) Ptf1acre/+;Ptch1fl/fl SHH-MB model to investigate BBB integrity. To assess the permeability of the BBB in mice at advanced stages of SHH-MB, symptomatic mice (14 weeks or older) were injected intravenously with tetramethylrhodamine (TMR)-dextran. We observed minimal extravasation of TMR-dextran into parenchymal brain tumour tissue, including following the administration of low-dose ionizing radiation (Extended Data Fig. 1). We conclude that the BBB of these mice appears to remain intact well into advanced tumour stages and thus parallels the physiology of human patients with SHH-MB6.
We next examined P-selectin expression in the SHH-MB tumour microenvironment and the effects of low-dose X-ray irradiation (XRT)18. We found that P-selectin is expressed in SHH-MB tumour vasculature in the absence of radiation (Fig. 1a) and that this expression could be further enhanced following a single 2 Gy dose of XRT (Fig. 1c). Notably, the elevation of P-selectin expression following whole-brain irradiation was confined to tumour regions and was not apparent in adjacent, normal brain tissue (Fig. 1d). To assess the potential to mitigate RT-related toxicity, we sought to identify the minimal required dose of irradiation that could still achieve robust induction of endothelial P-selectin expression. We found that P-selectin expression could still be robustly induced following a single dose of 0.25 Gy XRT (Fig. 1b and Extended Data Fig. 2). P-selectin expression was observed to reach substantially elevated levels at approximately 6 h following XRT, and these levels persisted for at least 24 h (Fig. 1e and Extended Data Fig. 3). To confirm the clinical relevance of endothelial P-selectin expression as a potential target molecule, we examined human SHH-MB tumour tissue surgically resected from paediatric patients. Immunohistochemical analysis similarly showed P-selectin expression in tumour-adjacent vasculature (Fig. 1f)….
