Innovative techniques for delivering therapeutic macromolecules to the brain cells is of considerable interest in biomedical research (Dong, 2018). Getting large molecules through blood brain barrier (BBB) for the treatment of traumatic brain injury still remains a great challenge for scientists. Modern new therapeutic methods fail as they rely on large molecular weight molecules delivery through BBB to the brain cells (Azarmi et al., 2020, Pardridge, 2012). Hence, finding ways to circumvent BBB and deliver macromolecules to the brain are strongly desired.

Naturally occurring nano sized exosomes are the best alternative drug delivery vehicles to the brain, because of their inherent cargo encapsulation and delivery properties (Ahmed et al., 2023, Khan et al., 2021). Exosome have recently been investigated in many studies as suitable substitutes for the shortcomings of traditional agents due to their biological compatibility and particularly small size for brain targeting (Terstappen et al., 2021). Hydrophobic and hydrophilic agents could be easily loaded to exosomes core either by simple mixing and incubation process or by physical stimulation of the exosome lipid bilayer membrane (Tenchov et al., 2022). However still some issues like limited disease site accumulation and low therapeutic outcome in the brain need to be addressed for their clinical translation (H. Ruan et al., 2023).

One of the major mechanisms for crossing the BBB is the receptor mediated endocytosis. Peptides legends specifically binds to the receptor present on BBB and provide highly effective features for brain targeting. RVG29 has inherent nicotinic acetylcholine receptor (nAChR) binding ability, which is abundantly present on neuron and endothelial cells of BBB (You et al., 2022). Previously RVG29 decorated exosomes were developed via genetically engineering exosomes producing cell and were used as delivery vehicle to the brain (Alvarez-Erviti et al., 2011, Kim et al., 2019). We engineered exosomes with RVG29 via bio-orthogonal click chemistry technique which is a simple, reliable, fast, and highly efficient technique for bioconjugation of small and macro molecules to the exosomes surface (Scinto et al., 2021).

Post synaptic density protein 95 is a promising therapeutic target for neuroprotection after brain injuries because it links the N-Methyl-D-aspartate receptor (NMDAR) to neurotoxic signaling pathways (Sattler et al., 1999). During brain injury the postsynaptic density protein 95 (PSD-95) binds to NMDARs and neuronal nitric oxide synthase (nNOS), forming NMDAR-PSD-95-nNOS death signaling complex. This complex leads to activation of nNOS by the calcium that fluxes through the NMDAR (Lai et al., 2014). Subsequently, this complex trigger the overproduction of nitic oxides, generating reactive oxygen species (ROS) and ultimately results in neuronal death (Ballarin and Tymianski, 2018). Efforts aimed at directly blocking or genetically deleting NMDARs have been shown to have detrimental effects leading to neuropathy and neuronal death (Ge et al., 2020). Therefore, promising approach could be disrupting the interaction of PSD-95 with NMDARs, which is possible by loading neurons with NR2B9C, a nine amino acid COOH-terminal residue of NMDARs (X. Yu et al., 2018). NR2B9c could prevent NMDAR-mediated neurotoxicity during brain injury without affecting essential NMDAR activity. Reducing the NMDAR mediated neurotoxicity during the secondary injury is one of the core therapeutic strategy during ischemic stroke and TBI (Parsons and Raymond, 2014). However, due to large size of NR2B9c, it can hardly enter the brain and neurons to exert its therapeutic effect because of the existence of BBB and the neuron membrane. Then attempt was made to modify NR2B9c with TAT, a peptide from HIV. Neuroprotection via Tat- NR2B9c has been enormously investigated after stroke in mice (Teves, Cui, and Tymianski, 2016), rats (Aarts et al., 2002, Sun et al., 2008), high order primates (Cook et al., 2012a, Cook et al., 2012b) and in human’s clinical trials (Hill et al., 2020, Hill et al., 2012). However, TAT peptide lacks specificity in targeting hence produce off-target effects. Moreover, TAT is unstable, have short half-life and its potential effects on human’s genes is poorly understood as well as it might break the BBB, therefore the safety of TAT is controversial (Kristensen et al., 2020, Papadopoulou and Tsiftsoglou, 2013).

In this study we sought to investigate the potential of NR2B9c loaded into click chemistry developed RVG29 decorated exosomes for the treatment of TBI (Scheme-1). We first introduced the azide group to RVG29 via the amidation reaction. Then, the exosomes were modified with dibenzocyclooctyne (DBCO) by reacting the amin group of exosome surface proteins and DBCO-terminated PEGylated N-hydroxysuccinimidyl ester (DBCO-PEG4-NHS). The functionalized exosomes were decorated with RVG29 via copper-free azide-alkyne cycloaddition reaction. Finally, the RVG29 decorated exosomes were loaded with NR2B9c via ultrasonication protocol generating RVG-ExoNR2B9C and its physical and functional properties were evaluated. Series of in vitro and in vivo studies were performed to investigate the neuron and brain targeting potential of exosomes formulations. In vitro oxygen glucose deprivation model and in vivo controlled cortical impact model of TBI were used for evaluation of the neuroprotective abilities of RVG-ExoNR2B9C. The RVG-ExoNR2B9C had no observable side effects on brain cells and organs in vivo. Overall, we demonstrate that the combination of naturally occurring nano sized exosomes as brain cargo delivering system, RVG29 as neuron targeting agent and NR2B9c as neuroprotective agent is a clinically practicable strategy for TBI treatment (see Fig. 1, Fig. 2).