Spray-dried L-cysteine modified chitosan microparticles encapsulate superfolder green fluorescent protein as a model for nose-to-brain drug delivery

Biopharmaceuticals are promising drugs in the treatment of many diseases, including degenerative and chronic diseases affecting the central nervous system (CNS), such as multiple sclerosis, Alzheimer's disease, and epilepsy. However, the delivery of larger proteins to the CNS remains a significant challenge owing to the restrictive nature of the blood-brain barrier (BBB). Nose-to-brain drug delivery has emerged as a promising, non-invasive route to bypass the BBB and directly target the CNS. Among the strategies explored, thiolated polymers, such as L-cysteine-modified chitosan (Cys-Chitosan), offer enhanced mucoadhesive and permeation properties. Nevertheless, thiolated chitosan has been poorly investigated as a matrix material for protein encapsulation through spray-drying and nasal administration, especially under neutral pH conditions, which represent a challenging condition for chitosan and thiols. In this study, we demonstrate that L-cysteine chitosan microparticles produced by spray-drying under physiological conditions can encapsulate superfolder GFP (sfGFP) with high efficiency, ensuring protein stability, sustained release, and mucosal interaction. Our results indicated that using thiolated chitosan as a matrix material allowed for high encapsulation efficiency and maintained protein activity. Release and permeation studies demonstrated delayed and controlled delivery of sfGFP compared to free protein, with kinetic modeling confirming the non-Fickian release behavior. Moreover, Fourier transform infrared (FTIR) spectroscopy analysis revealed specific biochemical interactions between the microparticles and the nasal epithelium, indicating mucoadhesive and biointeractive properties. These findings highlight the potential of spray-dried Cys-Chitosan microparticles as a promising platform for the non-invasive nose-to-brain delivery of biopharmaceuticals, combining stable protein encapsulation, controlled release, and enhanced mucosal retention.