Efficient Regulation Of Microglial Polarization Based On An Engineered Light-responsive Macrophage Delivery System For The Prevention And Treatment Of Inflammatory Depression

Background and objective:Depression is one of the leading causes of the global burden of disease,which affects more than 350 million people worldwide.Traditional antidepressants,the slow onset time（～four weeks）and limited efficacy（～30%of patients do not respond to at least two antidepressants）cannot meet the growing clinical demand for the treatment of depression.Therefore,it is of great importance and urgency to develop new antidepressants strategies.Accumulating evidence has indicated that inflammation plays an essential role in the pathogenesis of depression.Thus,targeting the central nervous system to reduce microglia activation-induced neuroinflammation is a promising new therapeutic strategy for depression.Notably,as one of the innate immune cells,macrophages play essential roles in inflammation and have colossal application potential in drug delivery.Initially,a UZPM photoresponsive system,Na YF₄:Yb,Tm（UCNP）@zeolitic-imidazolate framework（ZIF-8）-photoacid（PA）+melatonin（MT）,was synthesized,accomplished with poly-ethylene glycol（PEG）surface modification to improve biocompatibility,and could rapidly respond to near-infrared（NIR）light to release MT.This system was then encapsulated by hydroxylamine-labeled liposomes,which acted as a modifier to introduce UZPM into the cytoplasm of macrophages through membrane engineering.Aldehyde-modified CTLA-4 was used as a chimeric antigen receptor（CAR）targeting group to modify the surface of macrophages by aldehyde/hydroxylamine condensation to precisely target central M1-type microglia.In conclusion,to precisely target central inflammatory M1 microglia,we successfully synthesized a cell membrane vaccine-like system,CAR-M-UZPM.Importantly,it could also efficiently regulate the immune microenvironment,possess the therapeutic advantages of rapidly reducing the expression of pro-inflammatory factors and preventing inflammation-related depression.Methods:1.Here,the light-responsive anti-inflammatory system UZPM was synthesized,which could quickly release MT in response to 980 nm NIR.Aiming to improve the biocompatibility of the system,PEG was used to modify the surface of the synthesized UZPM.Subsequently,PEG-UZPM was introduced into macrophages by functionalized liposome fusion,and hydroxylamine groups were introduced on the cell surface at the same time.Finally,CTLA-4 was treated with coenzyme pyridoxal phosphate（PLP）to convert its N-terminal into aldehyde group,and the effective connection reaction between hydroxylamine and aldehyde group was used to anchor CTLA-4 as target groups on the cell surface2.In vitro,the performance and biosafety of CAR-M-UZPM were explored using drug release experiments,cytotoxicity,live and dead cell staining and other experiments.3.In vivo,the therapeutic performance and biosafety of CAR-M-UZPM were comprehensively evaluated through experiments such as brain targeting of materials,treatment of depressed mice,and immunohistochemical staining of important tissues and organs.Results:1.UCNP was synthesized following previously reported methods.UCNP was modified with PVP（MW=40,000）,which was observed by TEM and showed the particle size to be approximately 50 nm.The particle size of UCNP@ZIF-8 was about70 nm.Elemental mappings of UCNP@ZIF-8 also demonstrated the successful preparation.The UCNP@ZIF-8 was further confirmed by Fourier transform infrared（FTIR）spectra,showing characteristic peaks of PVP at 1311 and 1660 cm^-1,and the peaks of ZIF-8 at 1144 cm^–1 were due to C-N stretching.2.We then analyzed the effect of LPS on the morphological changes of BV2 cells by light microscope,and the results showed that MT inhibited the morphological transformation to classic short spindle shaped（M1 type microglia）after LPS stimulation.Furthermore,enzyme-linked immunosorbent assay（ELISA）results showed that MT dramatically suppressed the release of pro-inflammatory factors（Interleukin-4（IL-6）and TNF-α）,while promoting the secretion of anti-inflammatory factors（IL-4 and IL-10）.In addition,immunofluorescence staining results showed that MT inhibited the polarization of M1 type microglia as indicated by reduced expression of CD86,a pro-inflammatory marker.3.To further confirm the high permeation efficiency of CAR-M-UZPM,we then investigated the in vivo BBB penetration effect using the LPS injected mouse model.Compared with the UZPM and the M-UZPM group,CAR-M-UZPM could effectively cross the BBB within 6 h.Exhilaratingly,CAR-M-UZPM treatment dramatically reversed the changes of these behavioral parameters caused by LPS Our results indicated that CAR-M-UZPM treatment blocked the increased expression of the above-mentioned inflammation-related proteinsConclusion:In this study,a photoresponsive macrophage drug-carrying system was successfully constructed and employed as a vaccine-like inflammation-related depression prevention tool.This engineering drug delivery system could efficiently penetrate the BBB,accurately target the central M1 type microglia,and efficiently reduce the expression of central pro-inflammatory factors.More importantly,CAR-M-UZPM could precisely adjust the polarization type of microglia,promote the polarization of microglia to M2 type,and produce anti-inflammatory effects in the brain.Through depressive behavioral test,biochemical and neuro-electrophysiological analysis in vivo,it was proved that CAR-M-UZPM could prevent inflammation-related depression trigged by large doses of LPS.In short,the developed CAR-M-UZPM system with efficient central administration could overcome the complex physiological environment in the body and improve the efficiency and accuracy of central administration.