Quality by design (QbD) optimization of diazepam-loaded nanostructured lipid carriers (NLC) for nose-to-brain delivery: Toxicological effect of surface charge on human neuronal cells

Diazepam is commonly used in the management of epileptic seizures, although it has limitations that can be overcome by using formulations that are easier to administer and capable of directing the drug to the brain. In this field, it has been reported that the use of nanostructured lipid carriers (NLC) via intranasal (or via nose-tobrain) promotes the targeting of drugs to the brain, improving the effectiveness of therapy. The aim of this work was to optimize two diazepam-loaded NLC formulations for nose-to-brain delivery, one with positive surface charge and one with negative surface charge. The quality by design (QbD) approach was used to design the experiments, where the quality target product profile (QTPP), the risk assessment and the critical quality attributes (CQAs) were defined to ensure safety, efficacy and quality of the final formulations. The experiments started with the optimization of critical material attributes (CMAs), related to the ratios of lipids and emulsifiers, followed by the selection of critical process parameters (CPPs), related to the production methods of the diazepam-loaded NLC formulation (ultrasound technique and high-pressure homogenization - HPH). Afterwards, the positive surface charge of the diazepam-loaded NLC was optimized. Finally, the biocompatibility with human neuronal cells of the formulation with a negative surface charge and of the formulation with a positive surface charge was evaluated. The results of the optimization of the CMAs showed that the ratios of lipids and emulsifiers more adequate were 6.7:2.9 and 4.2:0.3 (% w,w), respectively. Regarding the CPPs, HPH was considered the most suitable production method, resulting in an optimized diazepam-loaded NLC formulation (F1C15) with negative surface charge, showing particle size of 69.59 +/- 0.22 nm, polydispersity index (PDI) of 0.19 +/- 0.00, zeta potential (ZP) of -23.50 +/- 0.24 mV and encapsulation efficiency (EE) of 96.60 +/- 0.03 %. The optimized diazepam-loaded NLC formulation (F2A8) with positive surface charge had particle size of 124.40 +/- 0.84 nm, PDI of 0.17 +/- 0.01, ZP of 32.60 +/- 1.13 mV and EE of 95.76 +/- 0.24 %. In addition, the incorporation of diazepam in NLC resulted in a sustained release of the drug. No significant changes in particle size, PDI, ZP and EE were observed for the formulation F1C15, after 3 months of storage, whereas for formulation F2A8, particle size increased significantly. Biocompatibility studies showed that the formulation F2A8 was more cytotoxic than the formulation F1C15. Thereby, we conclude that the formulation F1C15 is more suitable for targeting the brain, when compared with the formulation F2A8. From the results of these studies, it can be confirmed that the QbD approach is an adequate and central tool to optimize NLC formulations.

Automated summary

Optimized diazepam-loaded NLC formulation with negative surface charge showed smaller particle size, higher encapsulation efficiency, and better biocompatibility for targeting the brain compared to the formulation with positive surface charge. The sustained drug release effect was observed in both formulations, with the negative surface charge formulation exhibiting greater cytotoxicity than the positive surface charge formulation.