Neutrophil-mediated Puerarin Liposome Delivery System For Cerebral Ischemia-reperfusion Injury

Ischemic stroke is the leading cause of death and disability in the world.The current treatment methods are mainly through tissue-type plasminogen activator thrombolysis and surgical removal of blood clots to achieve blood flow reperfusion.However,CIRI（cerebral ischemia-reperfusion injury）is a new injury caused by blood circulation recovery after thrombolysis.Pueraria is a bioactive isoflavone-C-glucoside with a low oil/water partition coefficient.CIRI can cause ROS damage and inflammatory cascade,mainly involving BBB destruction,expression of cell adhesion molecules,and release of inflammatory mediators.As a base of p-glycoprotein transporter,after intravenous injection,it is extremely difficult to penetrate the blood-brain barrier（BBB）and cannot fully exert its anti-CIRI effect,which limits the clinical use of Pueraria in stroke-related diseases.Therefore,there is an urgent need to develop more effective targeted delivery methods to assist Puerarin to cross BBB and give full play to its advantages of anti-CIRI effect.At present,endogenous cell carriers have shown promise as effective targeted delivery systems to assist drug molecules to cross BBB.Neutrophils（NUS）are a kind of cells that enter BBB through chemotaxis of inflammatory cytokines and are also known as"small phagocytes"due to their phagocytosis of foreign bodies.As endogenous cells,NUS is endowed with inflammatory chemotaxis and cross-BBB ability.When the body is injured,NUS is drawn by the inflammatory chemokines at the injured site,and arrives at the injured tissue along with the blood circulation,killing bacteria by phagocytosis of pathogens.Also,the activation of Neutrophils stimulated by antigen leads to the migration of a variety of proteases into the NUS nucleus and the release of chromatin from the NUS nucleus to the outside of the NUS cell,forming Neutrophils extracellular traps（NETs）to phagocytosis and kill other pathogens and repair the damage.In this study,Puerarin was mediated by NUS as the carrier,drug molecules were actively targeted to deliver and cross BBB to the site of brain injury by taking advantage of the inflammatory environment of the brain after CIRI and the inflammatory and deformable properties of NUS,so as to give full play to the anti-CIRI effect of Puerarin.Firstly,Puerarin cationic liposome（L-Ps）was prepared to increase the drug loading and stability of Puerarin in NUS.The automatic"capture"of L-Ps by NUS was realized by charge absorption,and a neutrophil-loaded Puerarin cationic liposome targeted drug delivery system（NUS-L-Ps）was constructed.At the same time,the"proton sponge effect"of cationic liposomes ensured the intracellular stability of L-Ps after it was engulfed by NUS.Secondly,depending on the chemotaxis of NUS on inflammatory factors and the deformability of NUS itself,L-Ps can be effectively transported through BBB and delivered to the brain parenchyma,and Puerarin can be successfully transported to the damaged brain parenchyma.Third,NUS was used to release NETs responsively after NUS-L-Ps reached the site of brain injury.Finally,by targeting the damaged nerve cells with the positive charge on the surface of L-Ps again,Puerarin’s antioxidant neuroprotective effect was fully exerted.This topic will study the anti-CIRI effects of NUS-L-Ps from the following four parts:（1）The construction of NUS-L-Ps targeted drug delivery system;（2）Characterization of the properties of NUS-L-Ps targeting drug delivery system in vitro and study of the neuroprotective effect of Puerarin in vitro;（3）Study on drug delivery behavior and efficacy of NUS-L-Ps targeted drug delivery system in vivo;（4）Preliminary study on the mechanism of neuroprotective action of NUS-L-Ps targeting drug delivery system in vivo.（1）Pueraria cationic liposomal（L-Ps）was prepared by the thin-film dispersion method.The formulation and technological conditions for the preparation of L-Ps were screened and optimized by using average particle size,Zeta potential,drug loading efficiency（LE%）,and encapsulation rate（EE%）as evaluation indexes.The particle size of L-Ps was 107.26±0.55 nm,and the Zeta was 33.30±1.93 m V.Fluorescein Isothiocyanate（FITC）-activated Puerarin cationic liposomes（FITC-L-Ps）,common liposomes（Ls）and FITC-Ls were prepared by the same method.The liposomes prepared were in line with the requirements of the later experiment.Taking cell yield,cell survival rate,and cell purity as evaluation indexes,the extraction and separation methods of drug carrier NUS were compared and screened.The optimal extraction method of NUS was the PERCOLL gradient centrifugation method.The extraction rate was 6×106 cells/mouse,the survival rate was more than90%,and the purity was also more than 95%,which met the requirements of the drug carrier in the later stage.Using Puerarin content as the evaluation index,compared and evaluated the loading amounts of L-Ps and Ls in NUS,and it was proved that cationic liposome could improve the phagocytosis amount of Puerarin in NUS.The conditions of NUS loading L-Ps were further optimized,and the optimal construction conditions of NUS-L-Ps were selected as follows:the final concentration of L-Ps was 100 g/106 cells co-incubated with NUS for 1.5 h.Also,it was proved that the survival rate and morphology of NUS cells were not significantly changed after NUS phagocytosis of L-Ps,which initially verified the high biocompatibility and low toxicity of L-Ps.（2）The drug delivery behavior of NUS-L-Ps and the efficacy of Puerarin in vitro were studied.The in vitro properties of NUS-L-Ps were comprehensively characterized by the studies on the inflammatory activity,trans-BBB ability,release behavior,and uptake behavior of PC12 nerve cells in vitro.It was proved that NUS-L-Ps carried L-Ps stably through the blood circulation,actively targeted delivery,and through BBB to the damaged parts of the brain by using the inflammatory chemotaxis and deformability of NUS.After NUS-L-Ps reaches the brain-damaged site,L-Ps is localized and released,and the EE%and morphology of L-Ps released are the same as before phagocytosis.L-Ps cations can be used again to increase the uptake of Puerarin by the damaged target cells and give full play to the neuroprotective effect of Puerarin.PC12 nerve cells were damaged by L-glutamate as the model,and the in vitro pharmacokinetic study of Puerarin against L-glutamate injury of PC12 nerve cells was carried out using cell survival rate,cell morphology,and ROS oxidative stress as the evaluation indexes.It was proved that Puerarin had an obvious neuroprotective effect in vitro,and showed a certain correlation with Puerarin concentration.The preliminary study on the mechanism of efficacy in vitro proved that Puerarin could exert the protective effect of PC12 nerve cells by inhibiting ROS oxidative stress in nerve cells.（3）The drug delivery behavior and efficacy of NUS-L-Ps were studied in vivo.Firstly,the preparation method of the Middle cerebral artery occlusion（MCAO）model for this study was optimized.The survival rate of MCAO model mice prepared by this method is about 90%,and the neurological function score（Zea-Longa score）is between 3-4,which is suitable for the follow-up study of pharmacokinetics,tissue distribution,and in vivo efficacy.The pharmacokinetic results of the MCAO model mice showed that the cycle time of the NUS-L-Ps and L-Ps was longer,and with the extension of time,the clearance rate of the L-Ps group was significantly faster than that of the NUS-L-Ps.In addition to the longer blood retention rate,the area under the curve（AUC）and elimination half-life(T_1/2)of the NUS-L-Ps were also significantly better than the L-Ps.AUC_0-∞in the NUS-L-Ps was 1.41 times that in the L-Ps,and t_1/2was 1.54 times that in the L-Ps.In vivo,fluorescence imaging results of MCAO model mice showed that NUS-L-Ps had active targeting drug delivery behavior to brain damage sites,and further tissue quantitative determination results proved that NUS-L-Ps had more enrichment in brain damage sites.All the above studies proved that NUS-L-Ps could actively target the damaged brain tissue in MCAO model mice and improve the BBB transmission rate of Puerarin.Calculating the infarct area in vivo,it was found that both the NUS-L-Ps and the L-Ps showed certain advantages in drug administration,NUS-L-Ps was superior to L-Ps within 3 hours after surgery,while there was no significant difference between the NUS-L-Ps and the L-Ps after 6 h.Both L-Ps and NUS-L-Ps showed the advantages of medication through the spontaneous activity behavior study,and the NUS-L-Ps showed more motor ability and exploration behavior significantly.MWM studies show NUS-L-Ps set in the target quadrant percentage of swimming time than another control group,a significant increase in the residence and the target quadrant time,indicating that the mice had better memory ability after NUS-L-Ps administration.All the above studies proved that NUS-L-Ps fully played its anti-CIRI effect by increasing the amount of Puerarin entering the damaged brain parenchyma.（4）The neuroprotective mechanism of the NUS-L-Ps delivery system in vivo was studied.The study showed that the RNS clearance efficiency of the NUS-L-Ps was higher than that of the L-Ps.The total NO of NUS-L-Ps was reduced by 89.86%compared with the normal saline group,while the reduction rate of L-Ps was only41.36%.Similarly,the removal efficiency of ROS in the NUS-L-Ps was higher than that in the L-Ps.NUS-L-Ps is reduced ROS 58.08%,while that of L-Ps was only29.72%.It was proved that NUS-L-Ps could deliver Puerarin to the brain injury site by reducing the ROS and RNS levels in the focal area after MCAO operation and reducing the brain injury caused by reactive oxygen species and NO,to fully play the neuroprotective effect of antioxidant activity of Puerarin.In summary,a neutrophil-mediated Puerarin cationic liposome targeted drug delivery system（NUS-L-Ps）was constructed for an anti-CIRI study.DC-CHO-L was used to prepare Puerarin cationic liposomes to reduce the toxicity of preparations to carrier cells.Meanwhile,the charge absorption of cations was used to increase the load of NUS and the stability of Puerarin in the carrier of NUS.This idea of increasing the drug load of NUS was innovative.Using the inflammatory chemotaxis of NUS,cross-BBB,and release of NETs,we mediated the stable delivery of Puerarin through the blood circulation to the damaged brain parenchyma and through BBB,NUS-L-Ps reached the damaged brain site,and Nus-L-Ps was released in response to inflammation and released complete L-Ps.The positive charge of L-Ps was used again to increase the uptake of damaged target cells and give full play to the anti-CIRI effect of Puerarin.The brain-targeted delivery mechanism of cell-nano preparations of traditional Chinese medicine ingredients is innovative and has a good demonstration effect on both insoluble traditional Chinese medicine and chemical drug ingredients with large clinical effective doses,laying a solid foundation for the clinical application of cellular carrier in CIRI therapy.