Zonisamide nanodiamonds for brain targeting: A comprehensive study utilising in silico, in vitro, in vivo, and molecular investigation for successful nose-to-brain delivery for epilepsy management

The blood-brain barrier (BBB) is a stringent barrier that restricts the successful brain delivery of polar neurotherapeutics molecules. One such molecule is Zonisamide (ZNS), a hydrophilic centrally acting anti-epileptic drug. This study aims to overcome the poor ZNS BBB permeability using the nose-to-brain (NTB) carbon-based biocompatible nanodiamonds (ND) delivery system to deliver ZNS directly to the brain, bypassing the BBB, thereby enhancing its efficacy and reducing systemic side effects associated with oral ZNS currently available formulation in clinical practice. Intranasal (IN) ND-ZNS formulations were optimised using an Artificial neuronal network (ANN) and assessed for particle size (PS), zeta potential, loading efficiency (%LE), morphology, and in vitro release. The optimum radiolabelled ND-ZNS complex F1 biodistribution in different organs and its pharmacokinetics were compared to oral and IN-free ZNS in mice. Temporal lobe epilepsy (TLE) model in rats was used to compare the anti-epileptic activity of IN ND-ZNS F1 to IN free ZNS by assessing brain activity, epileptic biomarkers such as (brain neuronal specific enolase (NSE), neurofilament light polypeptide (NEFL), and matrix metallopeptidase-9 (MMP-9)), hippocampal histopathology and the modulatory effect on epigenetic miR-199/SIRT-1 and PVT-1/BDNF pathways. Optimized ND-ZNS complex F1 consists of a ZNS:ND ratio of 1:2 and sonicated for 5 min exhibited the least PS (193.7 ± 19.3 nm), adequate %LE (87.1 ± 9.2%) similar to ANN predictions, with a biphasic in vitro release profile of ZNS, beneficial for both acute and chronic epilepsy treatment. The IN delivery of ND-ZNS complex F1 showed preferential higher in vivo brain uptake with minimal systemic exposure linked with higher brain/blood ratio and significant (p ≤ 0.05) overall enhanced pharmacokinetics expressed by C_max and AUC _(0-120min) when compared to oral and IN free ZNS_. Moreover, the TLE model confirmed the improved anti-epileptic activity of F1 compared to IN-free ZNS regarding brain activity and hippocampal histopathology, significant suppression of serum NSE, NEFL, MMP-9 levels, miR-199/SIRT-1 pathway, and normalization of PVT-1/BDNF pathway. Therefore, ND used in this study could be a novel, promising carrier to target ZNS directly to the brain via the IN route for effective epilepsy management with less drug dosing and the least systemic side effects.