Mechanism Of The Brain Clearance Of Reconstituted High Density Lipoprotein Nanoparticulate Drug Delivery System

The development of nanotechnology has provided considerable strategies to facilitate brain drug delivery by overcoming the blood-brain barrier?BBB?.However,most researches at present mainly focus on the efficiency of brain drug delivery while little attention has been paid to the key issue that how the nanoparticulate drug delivery system?NDDS?be cleared from the central nervous system?CNS?.Considering the safety of drug application,it is also necessary to characterize the in-brain distribution,metabolism and excretion of NDDS to provide fundamental basis for the rational design and application of NDDS for brain-targeting drug delivery.Based on our perivous work,we hypothesized that the brain clearance of NDDS might not be only mediated by in-situ metabolism,and the recently identified glymphatic system could be another important clearance pathway.To test this hypothesis,we used ApoE-reconstituted high density lipoprotein?ApoE-rHDL?,which possesses the ability to target the core lesion in the brain of Alzheimer's Disease?AD?,as the model of brain-targeting NDDS.Two-photon fluorescent microscopy which possesses the advantages such as deep tissue penetration,high resolution and high sensitivity would be applied as a key technique for the in-situ visualization of brain vasculature,glymphatic system and brain cells,and for the real-time imaging of the in-brain transport and clearance of ApoE-rHDL.It was found that both astroglia and microglia internalized ApoE-rHDL efficiently.In addition,ApoE-rHDL showed the trend to accumulate in the paravascular glymphatic system.Radiolabeling was utilized to characterize the dynamics of the brain clearance of ApoE-rHDL.Relatively high radioactivity of ¹²⁵I-labeled ApoE-rDHL was found in the mouse cervical lymph nodes and the brain clearance of ¹²⁵I-labeled ApoE-rDHL was suppressed significantly after inhibiting the function of glymphatic system by the application of aquaporin-4?AQP-4?antibody.These results clearly suggested the important role of glymphatic system involved the in-brain clearance of ApoE-rHDL.Using the highly sensitive and selective liquid chromatography-mass spectrometry,we also characterized the degradation kinetics of ApoE-rHDL in glial cells in vitro.It was found that microglia showed a stronger ability to uptake ApoE-rHDL than astroglia while the degradation t_1/2 of ApoE-rHDL was similar in the two kinds of glial cells.At last,by comparing the difference in the brain clearance efficiency and intracellular degradation kinetics between ApoE-rHDL and monosialotertrahexosylganglioside?GM1?-modified ApoE-rHDL,we justified the concept that the physical and chemical properties of NDDS could affect their brain clearance efficiency.In summary,we here revealed the important role of glymphatic system and in-situ metabolism in the brain clearance of ApoE-rHDL by studying the brain clearance pathways,dynamic characteristic of ApoE-rHDL and the impact of pharmaceutical factors therein.We believe the findings of this work will provide fundamental basis for rational design and application of NDDS for brain-targeting drug delivery.