Preparation And Properties Of Redox-sensitive EGCG/Cys Cross-linked Drug-loaded Nanoparticles

Cardiovascular disease is a serious threat to human health and survival,and atherosclerosis is the common pathological basis for coronary artery disease,stroke and peripheral vascular disease.Therefore,people have to search for an effective atherosclerosis treatment nowadays.In recent years,nano drugs have become a research hotspot in the treatment of various diseases due to their targeting,enhancing drug water solubility,drug loading stability and diversity of drug releasing properties.Studies have shown that the atherosclerotic plaque is in an oxidative stress state caused by superoxide anion,hydroxyl radical,H₂O₂ and reactive oxygen species?ROS?,due to the redox state imbalance.The structures of the drug carriers changes occur in the oxidizing microenvironment to enable specific release of the drugs.In addition,atherosclerosis,as a chronic inflammatory disease,usually accompanied by hyperplasia.Rapamycin?Rapa?,a highly hydrophobic anti-rejection drug that acts on core regulators of cell homeostasis,which has anti-inflammatory,inhibits smooth muscle cell?SMC?proliferation and migration,promotes autophagy and immune regulation,and a variety of functions,with potential anti-atherosclerosis.In this paper,green tea extract epigallocatechin gallate?EGCG?and cystamine?Cys?were cross-linked in solution by Schiff base reaction and Michael addition reaction to form EGCG/Cys nanoparticles.In addition,rapamycin was nucleated in solution,EGCG and cystamine were cross-linked to form a shell to construct EGCG/Cys?Rapa?core-shell structure drug-loaded nanoparticles.For the other nanoparticles,rapamycin was loaded during the cross-linking process by solubilizing,and EGCG/Cys/Rapa uniformly dispersed structure drug-loaded nanoparticles were constructed.Since the disulfide bond in the cystamine molecule can break bonds in the atherosclerotic oxidative microenvironment state,a specific drug release function can be achieved by nanoparticle degradation.The nanoparticles were observed to be spherical by scanning electron microscopy?SEM?and transmission electron microscopy?TEM?.The EGCG/Cys nanoparticles were detected to have a particle size of about 100-300 nm using dynamic light scattering?DLS?,and the other two drug-loaded nanoparticles were about 50-200 nm.X-ray photoelectron spectroscopy?XPS?results showed the-C-N-and-C=N-groups in the crosslinked structure of nanoparticles,and it also proved that the degrades of the nanoparticles due to the breakage of disulfide bond and the loss of sulfur element after the redox treatment.Fourier transform infrared spectroscopy?FTIR?results also confirmed the chemical structure of the nanoparticles,and the successful loading of rapamycin in EGCG/Cys?Rapa?nanoparticles and EGCG/Cys/Rapa nanoparticles.The drug loading rates were determined by high performance liquid chromatography?HPLC?to be 24.381%and 7.379%,respectively.In vitro drug release results showed a faster drug release rate of EGCG/Cys?Rapa?nanoparticles in glutathione?GSH?solution compared to H₂O₂ solution,in 8 h.After 8 h,the total amount of drug released in H₂O₂ exceeded that in water.For EGCG/Cys/Rapa nanoparticles,the fastest drug release rate in 20 mM H₂O₂ solution resulted in a higher total drug release than that in GSH solution in 24 h.The release rate and total release amount in H₂O₂ solution or GSH solution were both higher than that in H₂O.Therefore,it could be stated that both drug-loaded nanoparticles have redox-responsive release characteristics.The presence of nanoparticles had a certain inhibitory effect on endothelial cells?ECs?,smooth muscle cells?SMCs?and macrophages.For EGCG/Cys?Rapa?and EGCG/Cys/Rapa drug-loaded nanoparticles,it was more obvious for the inhibition effect on SMCs due to the loading of rapamycin.Cellular uptake behavior of nanoparticles exhibited time-dependent and concentration-dependent.In addition,the three nanoparticles could significantly inhibit the secretion of the inflammatory factor interleukin-6?IL-6?and tumor necrosis factor-??TNF-??by macrophages at a very low concentration.The hemolysis rate of the three different concentrations of nanoparticles was less than5%,which ensures the safety of the nanoparticles in the blood circulation;the acute toxicity assessment of each major organ also showed certain biosecurity of the three nanoparticles,which were not enriched in tissue,and caused inflammation.In this paper,the REDOX-sensitive EGCG/Cys cross-linked nanocarriers provide a loading method for the strong hydrophobic drug Rapa.It is desirable to achieve a personalized drug release function,which depending on the oxidation degree of the pathological environment in atherosclerosis.It could effectively reduce the adverse reactions associated with oral drugs and improve the bioavailability of drugs in the body.The nanoparticles provide new ideas for the treatment strategy of atherosclerosis.