| Glioblastoma(GBM)is the most invasive and fatal primary brain tumor with a high mortality rate.Traditional tumor resection schemes are also limited due to the special location of glioma,and postoperative often results in poor prognosis due to tumor cell invasion.At the same time,the blood-brain barrier(BBB),as an important physiological barrier,prevents toxic substances from invading the brain while preventing most drugs from entering the brain.Therefore,the design of safe carriers that can help drugs to efficiently penetrate the BBB has become a hotspot in glioma research.Therefore,more and more drug delivery systems for tumor inflammation microenvironment have been developed,such as using immune cells as drug delivery carriers,but chemotherapy drugs often have certain toxicity to cells,and immune cells play a multifaceted role in the tumor microenvironment,which makes use immune cells directly as drug carriers do challenging.Exosomes(Exosomes,Exos)has attracted widespread attention as drug delivery vehicles due to their nano-effects,immunological properties,long-range targeting,and their ability to naturally penetrate physiological barriers.Studies have shown that cell-derived Exos often has the same components or similar biological functions as progenitor cells.Neutrophils(NEs)have been used for drug delivery targeting tumor microenvironments due to their ability to respond quickly to inflammation and have the effect of naturally penetrating BBB.In this study,we extracted Exos from NEs and characterized NEs-Exos.Next,the ability of NEs-Exos to target tumor microenvironment through BBB and respond to chemotaxis peptides and inflammation was be explored.Doxorubicin(DOX)was used as an antitumor model drug,and the effect of NEs-Exos/DOX on glioma was evaluated in vivo by using a glioma orthotopic transplantation model.This research helps to provide new options for targeted drug delivery in non-invasive inflammatory microenvironments for glioma and other central nervous system(CNS)diseases,as well as provides a new opportunity to explore additional endogenous vector tools based on Exos characteristics.The main research contents and corresponding results of this topic are as follows:(1)Extraction and characterization of NEs:NEs were isolated from mouse bone marrow,and cell purity was determined.The results showed that Ly-6G~+CD11b~+positive cells in the extracted account for 89.24%.Wright’s staining showed that the cells conformed to the morphology of NEs.(2)Characterization of NEs-Exos and NEs-Exos/DOX:NTA and TEM characterizations show that NEs-Exos appears as a round cup or saucer-shaped membrane bubble of uneven size with a peak particle size of 99.5±2.0 nm.WB indicates that NEs-Exos is rich in Exos-related proteins(TSG101,Alix and CD63).The Zeta potential of NEs-Exos is-11.80±1.31 m V,which proves that we have successfully extracted NEs-Exos.After NEs-Exos was loaded with anti-cancer drug DOX,fluorescence images showed that NEs-Exos/PKH67(green)and DOX(red)were co-localized(yellow),while NTA and TEM showed that the peak particle size of NEs-Exos/DOX became larger(134.0±10 nm),the morphology showed more heterogeneity.These results indicate that NEs-Exos has the ability to encapsulate drugs.The drug loading rate of NEs-Exos is 6.51%,and the encapsulation rate is 13.71%.The Zeta potential of NEs-Exos/DOX did not change significantly(-9.47±0.46 m V).(3)NEs-Exos can be uptaken by cells:NEs-Exos labeled by PKH67.And NEs-Exos/PKH67 can be taken up by mouse brain microvascular endothelial cells(b End.3)and rat glioma(C6)cells.Over time,the fluorescence intensity of NEs-Exos in cells gradually increases,and C6 cells showed strong uptake capacity.Cells were treated with various inhibitors such as colchicine,chlorpromazine,filipepine,brefeldin A,and chloroquine respectively,and internalization experiments were performed.It was found that the clathrin endocytosis pathway influenced by chloroquine mainly affected NEs-Exos/PKH67 cell internalization.(4)NEs-Exos responds to chemotaxis peptides and penetrate the BBB in vitro:The Transwell model was used to simulate BBB in vitro and explore the NEs-Exos response to chemotaxis peptides(f MLP).It was found that under the condition of f MLP,the accumulation of NEs-Exos/DOX at the bottom of the cell for 12 h increased by 1.88times to 36.24 ng(calculated as DOX)compared with that without f MLP.Similarly,under f MLP conditions,the fluorescence intensity of NEs-Exos/PKH67 gradually increased in C6 cells,indicating that NEs-Exos has a similar chemotactic ability as NEs,can effectively respond to chemotaxis peptides,and penetrates in vitro BBB.(5)Evaluation of NEs-Exos response to inflammation:LPS was used to establish an in vivo and in vitro inflammation model.After LPS treatment of C6 cells and b End.3cells,the uptake of NEs-Exos/PKH67 by the cells was significantly increased compared with the untreated group.The brain sections of LPS-injured animal models showed inflammatory cell infiltration,increased levels of pro-inflammatory factor(TNF-α),decreased expression of anti-inflammatory factor(IL-10),and increased expression of IBa1 and GFAP.Brain slices from the administration group after LPS injury showed that the expression of IBa1 and GFAP increased and accompanied with the accumulation of more NEs-Exos/PKH26 in brain.The results were more clearly observed in the enlarged fluorescent image,indicating that inflammation leads to chemotactic movement of NEs-Exos/PKH26.(6)NEs-Exos targets the tumor microenvironment in vivo and penetrates BBB:Zebrafish experiments show that compared to free DOX,NEs-Exos/DOX can leak out of blood vessels in the brain and effectively deliver drugs to zebrafish brain tissue.In vivo distribution experiments of tumor-bearing mice show that NEs-Exos/Di R loaded with Di R can penetrate BBB and target brain gliomas of tumor-bearing mice compared with free Di R.In vitro imaging further showed that NEs-Exos/Di R accumulates mainly in brain glioma regions.Immunofluorescence of the brain slices at 24 h showed that the tumor had a pathological environment with high IBa1 expression and low GFAP expression,and NEs-Exos/Di R accumulated in areas that with high expression ofα-SMA.This phenomenon was not observed locally in normal brain tissue,indicating that NEs-Exos/Di R can respond to the glioma inflammatory microenvironment,thereby targeting accumulate in the glioma area.(7)In vivo pharmacodynamic experiments:After NEs-Exos/DOX treatment,the fluorescence intensity of C6-Luc cells in the brain of tumor-bearing mice increase slowly compared to the saline and DOX groups,and brain tumor growth was significantly inhibited.The survival curve also showed that the survival time of the NEs-Exos/DOX group was significantly higher than that of the control group,with a median survival period of 27 days.H&E staining of brain sections showed that glioma cells were sparsely arranged in the NEs-Exos/DOX group.At the same time,immunohistochemical results showed that Ki-67 expression was lower in NEs-Exos/DOX group compared to DOX group and saline group,while GFAP expression was relatively high,indicating that NEs-Exos/DOX can effectively inhibit the malignant proliferation of tumor cells and reduce the malignant biological behavior of gliomas. |
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