Monocyte-based Targeted Delivery Of Bindarit For Prevention Of Atherosclerosis

Atherosclerosis（AS）is a high risk factor leading to fatal coronary heart disease,stroke,peripheral vascular disease and other diseases.As a chronic inflammatory disease,studies have shown that the development process of AS is closely related to Monocyte chemotactic protein-1（MCP-1）.In the early stage of atherosclerosis,monocytes migrate into the arterial intima and become macrophages under the control of MCP-1.Macrophages adhere to vascular endothelial cells and migrate under the endothelium to take up lipids and transform them into foam cells,thereby forming plaques.The accumulation of a large number of macrophages in the plaque is the main sign of active inflammation.Therefore,inhibiting the secretion of MCP-1 to inhibit the migration and infiltration of monocytes is expected to be an effective strategy for early treatment of atherosclerosis.Bindarit（BIN）has been proven to be an effective selective oral inhibitor of MCP-1/CC chemokine CC motif ligand 2（CCL2）,but it still has oral formulation disadvantages.The drug retention rate in the lesions caused by specific distribution is low,and long-term,high-dose administration is required.Therefore,how to achieve targeted delivery of BIN to AS plaques through oral administration to reduce the dose and increase the concentration of drug retention in the lesion is the core issue of our research.After a preliminary research,our group found that through the bionic approach,the yeast capsules（YCs）with the contents removed can be targeted to the tumor site and acute inflammation site through the oral route.Therefore,in this project,based on similar principles,imitating the way bacteria infect the body through the intestine,using yeast capsules as a carrier,an oral BIN targeted delivery system for the treatment of AS was constructed.Method1.Preparation of yeast capsulesA certain amount of yeast was taken,washed twice with isopropanol after acid-base treatment,and then dried to obtain blank yeast capsules.2.Preparation and characterization of bindarit nanoparticlesA certain amount of bindarit（BIN）and polythene imide（PEI）was weighed and dissolved in dimethyl sulfoxide（DMSO）,then put into dialysis bag.The Bindarit/polythene imide（BIN/PEI）nanoparticles were obtained by dialysis in pure water for 8 hours.The particle size and surface potential were measured by a Nano Zetasizer（Nano ZS）and the morphology of the nanoparticles was observed by a transmission electron microscope.（TEM）.3.Preparation and characterization of yeast capsules loaded bindarit nanoparticlesA certain amount of YC was weighed and BIN/PEI NPs was added,and the BIN/PEI mass ratio was 1:1.The two were mixed evenly,the mass ratio of YC and（bin-PEI）was 1:1,and the mixture was incubated at 37℃for 3h.After freeze-drying,the yeast capsules loaded with bindarit nanoparticles were obtained,and the drug loading was measured by high performance liquid chromatography.The transmission electron microscope and laser confocal microscope were used to observe the embedding of the nanoparticles in the yeast capsules.4.In vitro release evaluation of yeast capsules loaded bindarit nanoparticlesPrepare a p H 7.4 phosphate buffer solution,mix the drug-loaded yeast capsules into physiological saline（PBS）,and take the supernatant at 9 time points（1 h,2 h,4 h,8 h,10 h,12h,24 h,36 h,48 h）.The release degree of BIN was determined by high performance liquid chromatography,the cumulative release percentage was calculated,and the cumulative release curve of drug was plotted.5.Study on the targeting effect of oral yeast capsules on atherosclerotic plaqueThe fluorescent cyanine dye 5.5（Cyanine 5.5,Cy5.5）is chemically bonded to the surface of the PEI through a chemical reaction,and the Cy5.5-PEI nanoparticles（CY5.5-PEI NPs）are prepared by dialysis in pure water against light.Repeat the above BIN and PEI synthesis steps to obtain BIN/PEI-Cy5.5 NPs.Then YC was incubated with BIN/PEI-Cy5.5 NPs to obtain fluorescently labeled yeast capsules（BIN-Cy5.5/YCs）.Apolipoprotein E-deficient mice(Apo E^-/-)were given a high-fat diet to establish an atherosclerosis model,and were given Cy5.5-PEI NPs and BIN-Cy5.5/YCs by gavage for 4 consecutive days.After euthanasia,the aorta and major organ tissues were stripped,and the fluorescence intensity was determined by a Living Imaging system.6.Pharmacodynamic evaluation of oral drug-loaded yeast capsules in the treatment of atherosclerosisApo E^-/-mice were used to establish an AS model.They were given saline,BIN,BIN NPs,and BIN/YCs,and gavage every three days.After 1 month of treatment,other major organs were dissected and the aorta was removed.Stain with Oil Red O to evaluate the development of plaque.Immunohistochemical staining was used to observe the distribution of plaques in mouse aortic root sections by CLSM.The proportion of mononuclear macrophages in the whole blood of mice was determined by flow cytometry.7.Preliminary safety study on the treatment of atherosclerosis with drug-loaded yeast capsulesAfter hematoxylin-eosin staining,observe the structure of organs and gastrointestinal tissues of each group of mice pathological changes.After hematoxylin-eosin staining,the structure of organs and gastrointestinal tissues in each group of mice pathological changes were observed.Result:The yeast capsules whose outer wall is mainly composed ofβ-1,3-D-glucan are successfully obtained after acid-base treatment based on yeast.And using the electrostatic interaction between the positively charged nanoparticles and the yeast capsules,they gather on the surface of the yeast capsules and then enter the yeast capsules to form BIN/YCs.After assembling polyethyleneimine and bindarit to form nanoparticles,the results of particle size analyzer and potential analyzer show that the size of the nanoparticles is uniform,with an average particle size of 25.24±2.62 nm and a Zeta potential of 34.70±0.30 m V.Using static electricity-mediated spontaneous deposition,through a simple suspension process,the BIN/PEI positively charged nanoparticles were successfully loaded into YC.TEM observation results showed that the nanoparticles were dispersed inside the yeast capsules.The average particle size is 3451.45±443.45 nm,and the Zeta potential is 0.01±0.02 m V.The positively charged bindarit nanoparticles and yeast-loaded bindarit nanoparticles are completely released within 8hours at p H 7.4.In vivo imaging results showed that after oral administration of BIN-Cy5.5/YCs,the accumulation of Cy5.5 in atherosclerotic plaques can be significantly increased.The analysis of the fluorescence intensity of various organs and tissues showed that the fluorescence intensity of lymph-related mesenteric lymph nodes,small intestine and aortic plaques all have a significant increase.The results of oil red O staining showed that the proportion of AS plaques in Apo E^-/-mice was significantly reduced after oral administration of BIN/YCs,and the level of related pro-inflammatory factors in the serum was significantly reduced.The preliminary safety evaluation showed that there were no significant differences in blood physiological indicators,liver and kidney functions,and no obvious pathological changes in organs and tissues.Conclusion:Using electrostatic adsorption,positively charged bindarit nanoparticles can be successfully loaded inside YC.After oral administration,BIN/YCs can be targeted to atherosclerotic plaques in mice,and this process is closely related to the lymphatic system and intestinal tissues.At the same time,mononuclear macrophages may play an important role in the targeting process.Oral BIN/YCs can effectively reduce the growth of AS plaques without obvious side effects.To sum up,this project constructed an oral targeted BIN delivery system for treating AS by constructing oral targeted agents to the inflammatory site and using yeast microcapsules AS drug carrier,which is expected to provide theoretical and experimental basis for the development of a new type of AS drug.