## 4 Discussion
The concentration-dependency experimental results of AP-BL and BL-AP both showed that the number of substances transported by NMN was concentration-dependent, and the higher the concentration, the higher the number of substances transported by NMN. The results of the transport percentage indicate that, when the concentration of NMN is greater than 0.5 mmol/L, the change in transport percentage is small, indicating that saturation has been reached when 0.5 mmol/L is administered. Therefore, the subsequent experimental administration concentration should be less than 0.5 mmol/L. This result further proves that the transmembrane transport mode of NMN is passive transport relying solely on concentration differences. Given that the transport capacity of NMN was moderate, we modified the dosage form of NMN to prepare liposomes coupled with targeted peptides to enhance the transmembrane transport capability of NMN. After the successful preparation of liposomes, the particle size of KB-lips and NMN-lips is mainly concentrated around 100 nm, while the particle size of ANG-NMN-lips and RVG-NMN-lips is mainly concentrated around 130 nm. Nanoformulations with a particle size range of 100-200 nm are more suitable for intravenous administration, which can reduce the absorption of phagocytic cells and improve drug exposure time by prolonging blood circulation time [35]. The liposomes prepared in this study have suitable particle sizes and meet the requirements for intravenous injection, providing the possibility of developing NMN as an injection. Comparative experiments on the transport capacity of NMN liposomes and NMN showed that, compared with NMN, the \(P_{app}\) ratio of AP-BL increased after coupling with targeted peptides, while the \(P_{app}\) ratio of BL-AP decreased. The \(P_{app}\) values of NMN-lips and NMN are relatively similar, indicating that the transmittance of NMN cannot be increased. After coupling with the ANG peptide and RVG peptide, only ANG-NMN-lips increased the permeability coefficient of NMN to \(10^{-5}\), and compared with the \(P_{app}\) value of NMN, ANG-NMN-lips extremely significantly improved the transport capacity of NMN, while RVG-NMN-lips significantly improved the transport capacity of NMN, indicating that ANG-NMN-lips makes it easier for NMN to pass through the BBB and increase its drug content in the brain, providing the theoretical basis for subsequent clinical studies. Recent research has shown that developing a biocompatible polydopamine (PDA) platform and covalently attaching lactoferrin ligands to PDA spheres can improve the BBB transport efficiency of NMN through the exocytosis transmembrane pathway [36]. Compared with this study, the results of both experiments indicate that NMN can pass through the BBB and increase its ability to cross the BBB. The difference is that one is an in vivo experiment and the other is an in vitro experiment. This experiment not only has the advantages of simple and time-saving preparation methods but also has the advantage of being able to load larger amounts of drugs into the body and target the brain. Previous studies have confirmed that NMN is specifically transported into small intestinal cells by Slc12a8 transporters, which increase NAD+ levels in various organs and tissues of the body as they circulate through the bloodstream [37]. However, NMN is prone to degradation after reaching a certain level of moisture and temperature. At present, most NMNs on the market are capsules and tablets. After taking NMN capsules or tablets, most of them are degraded in the stomach, and only a small portion of NMN reaches the small intestine. The results of this study indicate that making NMN into an injection, transporting it directly to the brain through the blood, and improving the ability of NMN to pass through the BBB and delaying its release rate through NMN-loaded liposomes coupled with targeted peptides reduces NMN loss and provides the possibility of acting on the brain to a greater extent.