Intracalvariosseous injection: an approach for central nervous system drug delivery through skull bone marrow with a preclinical research in stroke

BACKGROUND: Central nervous system (CNS) accessibility constitutes a major hurdle for drug development to treat neurological diseases. Existing drug delivery methods rely on breaking the blood-brain barrier (BBB) for drugs to penetrate the CNS. Researchers have discovered natural microchannels between the skull bone marrow and the dura mater, providing a pathway for drug delivery through the skull bone marrow. However, there has been no research on the feasibility, safety, and efficacy of this delivery method for drug treatment of stroke. METHODS: We used a microporous method for intracalvariosseous (ICO) injection to deliver drugs directly into brain parenchyma through skull bone marrow. Safety of ICO was assessed by monitoring changes in skull and peripheral blood neutrophil counts, and FITC-dextran extravasation across the BBB. Drug delivery pathways were observed through transparent skull-dura mater-brain tissue. In a rodent stroke model, NA-1 or Y-3 neuroprotective agents were administered via ICO to evaluate safety and efficacy by assessing neurological deficits, infarct size, neuroinflammatory factors, neuronal apoptosis, and liver/kidney function. Drug concentration in tissues was measured using fluorescence tracing and high-performance liquid chromatography to gauge ICO delivery efficiency. FINDINGS: ICO injection delivers drugs to the brain parenchyma through microchannels between the skull bone marrow and the dura mater, offering higher delivery efficiency than intravenous injection. After ICO injection, there were no changes in neutrophil counts in the skull bone marrow and peripheral blood, and the amount of FITC-dextran passing through the BBB remained unchanged. This confirmed that ICO injection does not cause skull infection or break BBB, which suggested ICO injection is safe and feasible. In the treatment of stroke with neuroprotective agents, although the drug dosage of ICO injection was lower than intravenous injection, drug accumulation in the brain increased after ICO injection, which helped repair nerve damage, reduce neuronal apoptosis, and decrease the expression of inflammatory factors. INTERPRETATION: ICO injection is a central nervous system drug delivery method that utilizes natural microchannels between the skull and dura mater for efficient drug delivery. Our results assessed the feasibility and safety of ICO injection at the preclinical level and evaluated its efficacy in animal models of stroke. The findings provided a foundation for the clinical translation of ICO injection. FUNDING: This study was supported by The National Natural Science Foundation of China (No. 82425101); Beijing Municipal Science & Technology Commission (No. Z231100004823036); Capital's Funds for Health Improvement and Research (2022-2-2045); National Key Research & Development Program of China (2022YFF1501500, 2022YFF1501501, 2022YFF1501502, 2022YFF1501503, 2022YFF1501504, 2022YFF1501505).