## 4 Discussion  
Drug delivery to a specific site of action is the key to the effectiveness of the drug, and the biological barrier during drug delivery may prevent the accumulation of drugs at the site of disease, thus limiting the effective response to drug treatment. The blood-brain barrier is one of the biological barriers of the human body. The blood-brain barrier can prevent some substances (mostly harmful) from entering the brain tissue from the blood. Drugs need to cross the blood-brain barrier to exert their therapeutic effects and treat brain diseases. NMN is widely present in natural animals and plants and has a positive effect on delaying aging and inhibiting the development of brain diseases. At present, there have been extensive studies on NMN in the academic community, but they have mainly focused on component analysis, quality control, and pharmacological activity research. There is still a blank in the research on crossing the BBB and the transport mechanism of NMN. Therefore, this study conducted a preliminary investigation into the transport mechanism of NMN and attempted to explore ways to increase NMN's passage through the blood-brain barrier. There are two methods for evaluating drug properties across the blood-brain barrier: in vivo and in vitro experiments. Compared with in vivo experiments, in vitro experiments have shorter experimental cycles and lower costs and can be used for high-throughput screening and to identify and study individual molecular mechanisms of blood-brain barrier transport [30]. We constructed an in vitro cell monolayer model evaluating across the BBB using hCMEC/D3 cells and proved the success of the cell monolayer model's establishment through two methods. A drug substance is considered to be poorly absorbed (0-20%) when \(P_{app}\) is lower than 1 \(\times\) 10\({}^{-6}\), moderately absorbed (20-70%) when \(P_{app}\) is between 1 and 10 \(\times\) 10\({}^{-6}\), and well absorbed (70-100%) when \(P_{app}\) is higher than 10 \(\times\) 10\({}^{-6}\)[31]. We investigated the capability and mechanism of NMN across the BBB using the cell monolayer model. The results of the transport and efflux experiments of NMN showed that the \(P_{app}\) value was between 1 and 10 \(\times\) 10\({}^{-6}\), indicating that the transmembrane transport ability of NMN is moderate. The main pathways for drugs to cross the BBB are passive transport, active transport, and endocytosis. Passive transport involves the transport from the bloodstream to the brain through hydrophilic pores on the cell membrane, known as paracellular pathways, under the influence of concentration differences. Active transport includes both transporter protein and receptor-mediated pathways, in which peptides and proteins bind to specific receptors, initiating the internalization of ligands and receptors into cells [16; 32; 33]. An important difference between these two pathways is that active transport routes mostly require the participation and support of carriers and motivation. Studies have shown that there is no significant difference in the \(P_{app}\) ratio between A-B/B-A and B-A/A-B, and it is less than 2, indicating that the two directions of transport are not concentration-dependent or saturated, indicating pure passive transport [34]. In this study, we found that the ratio of \(P_{app}\) values of NMN is about one, with no signs of carrier participation, thus we preliminarily inferred that the transport mechanism of NMN is passive transport. On this basis, time- and concentration-dependency bidirectional transmembrane transport experiments with NMN were conducted. The time-dependency experimental results of AP-BL and BL-AP both showed a trend of first increasing and then stabilizing at the receiving end, indicating that saturation has been reached, and 120 min is the optimal administration time.