Biomimicking Magnetic Nanocarriers Loaded With Andrographolide For The Treatment Of Endotoxemia

Endotoxemia is a common and complicated problem in modern clinical medicine.The pathogenesis of endotoxemia is generally caused by the invasion of endotoxin into blood circulation with a series of clinlcal symptom such as inflammatory response and microcirculatory disturbance.endotoxin is released from the cell wall of Gram-negative bacteria after the killing or apoptosis of it at the lesion site.Bacterial endotoxin is also called lipopolysaccharide?LPS?,which is composed of lipids and polysaccharides in molecular structure.The stability of LPS structure makes it impossible to remove by conventional sterilization methods.At present,the treatment of endotoxemia mainly focuses on antagonizing the inflammatory reaction or microcirculation disorders caused by LPS.The pathogenesis of endotoxin is generally considered to be the combination of LPS with the LPS receptor protein on the surface of immune cells,such as macrophages or mononuclear cells in the body,and then to stimulate the inflammatory pathway of immune cells and to produce a large number of inflammatory factors.With massive LPS stimulation,a waterfall inflammatory response syndrome can occur,leading to a series of clinical symptoms.In view of this mechanism,we designed a core-shell biobiomimetic drug delivery system?Fe₃O₄@MMs?by extracting macrophage cell membrane and enclosing it on the surface of Fe₃O₄ nanoparticles.Due to the inherited surface properties of macrophage membranes,Fe₃O₄@MMs can specifically bind to LPS molecules in vivo and in vitro,make the LPS molecules inactivated and to remove LPS molecules.In addition,Fe₃O₄ as a substrate to provide stability can also be used for magnetic targeting therapy.Finally,on the basis of the detain of LPS molecules,we loaded the anti-inflammatory drug andrographolide?AD?in the hydrophobic layer of the Fe₃O₄@MMs to constitute the Fe₃O₄@MMs@AD drug delivery system.When the LPS is interacted with Fe₃O₄@MMs@AD,the drug is rapidly released due to the insertion of LPS to the bilayer of Fe₃O₄@MMs,which achieves the purpose of the combined treatment of LPS-induced inflammation.In the second chapter,the positively charged Fe₃O₄ was prepared by solvothermal method,Macrophage cell membrane was extracted by differential centrifugation.And then The Fe₃O₄ and cell membrane was fused by the electrostatic action and coextrusion using a liposome extrusion apparatus to form the Fe₃O₄@MMs,and then the Fe₃O₄@MMs@AD drug delivery system was constructed through loading AD.Transmission electron microscopy and particle size distribution characterization showed that the membrane-coated nano drug delivery system Fe₃O₄@MMs was successfully constructed with a clear core-shell structure in a diameter of 110 nm.The surface Zeta potential of Fe₃O₄@MMs is similar to that of the bare cell membrane at-18 mV.The loading of AD showed that the encapsulation efficiency and drug load of AD were 84%and 7.2%,respectively,when the AD/Fe₃O₄@MMs was 1/10 and the reaction temperature was 37°C.In vitro release studies have shown that when LPS is present,the AD release can be facilitated.In the third chapter,the biosafety and pharmacodynamic effects of Fe₃O₄@MMs were verified by in vitro experiments.The cytotoxicity assay showed that the Fe₃O₄@MMs has no cytotoxicity and has a good biocompatibility.The results of in vitro adsorption experiments showed that Fe₃O₄@MMs can effectively reduce the concentration of LPS in the solution system at 37°C for 90 min.The detoxification of LPS by the combined treatment of free AD,Fe₃O₄@MMs and Fe₃O₄@MMs@AD was verified in cell experiments.The results showed that Fe₃O₄@MMs could effectively inhibit the LPS-induced cell death and reduce the production of reactive oxygen species?ROS?in cells,and the Fe₃O₄@MMs@AD combination therapy showed the best therapeutic effect.In the fourth chapter,the injection dose of LPS was determined by mouse experiment to establish the model of endotoxemia in mice.The results of animal experiments show that the serum levels of inflammatory cytokines such as interleukin6?IL-6?and tumor necrosis factor??TNF-??in endotoxin mice can be effectively reduced through the treatment of Fe₃O₄@MMs@AD,and the malondialdehyde?MDA?and superoxide dismutase?SOD?levels were close to normal levels.In vivo experiments show that the treatment effect of Fe₃O₄@MMs@AD in endotoxemia mice was better than AD or Fe₃O₄@MMs alone.Biodistribution studies showed that Fe₃O₄@MMs was mainly concentrated in liver and lung tissues.The results of liver and lung tissue sections analysis showed that the mice treated with Fe₃O₄@MMs@AD showed a slight damage in LPS poisoned mice,and with a lower mortality.