Construction Of Nucleic Acid/statins Co-delivery Yeast Microcapsule For The Oral Targeting Treatment Of Atherosclerosis

Cardiovascular disease is the most common cause of death worldwide.About 50%of cardiovascular diseases related deaths are caused by coronary artery disease.Atherosclerosis（AS）is the pathophysiological basis of coronary artery disease and the most important pathological process in the development of cardiovascular disease.The theory of lipid metabolism imbalance of macrophages has became a consensus of AS pathophysiology.Macrophages phagocytize oxidized low-density lipoprotein when the lipid load is too heavy,so that cholesterol accumulates abnormally in macrophages and is finally transformed into foam cells.Foam cells undergo apoptosis and necrosis,release lots of inflammatory factors and cholesterol crystals into the micro environment,and form the necrotic core.Therefore,it is of great significance in the prevention and treatment of AS to inhibit the eccesss accumulation of cholesterol in macrophages,reduce the foam formation of macrophages and the lipid deposition on the inner wall of blood vessels.At present,the therapy on AS mainly focus on two aspects:inhibition of cholesterol intake and promotion of cholesterol efflux.The process of intracellular cholesterol efflux is also known as the reverse cholesterol transport（RCT）of macrophages.In the RCT pathway,ATP binding cassette transporter A1（ABCA1）and ATP binding cassette transporter G1（ABCG1）on the membrane surface of macrophages present intracellular cholesterol to apolipoprotein A1（apoA1）,an extracellular cholesterol receptor,thus generates high-density lipoprotein and metabolizes it through the liver.Therefore,ABCA1 and ABCG1 play an important role in the process of RCT and the regulation of cellular lipid homeostasis.Upregulating the expression of ABCA1 and ABCG1can promote RCT,alleviate or even reverse the transformation of macrophages into foam cells,so as to achieve the goal of treating AS.MicroRNA33（miR-33）is a microRNA embedded in the intron of the host gene of sterol regulatory element binding protein（SREBP）,which can target ABCA1 and ABCG1 and inhibit their expression,and is considered to be the key regulatory factor of atherosclerosis.In addition,miR-33 has been found to regulate macrophage polarization,fatty acid oxidation and macrophage autophagy.Previous studies have shown that silencing miR-33 can slow the progression of AS in ApoE knockout(ApoE^-/-)mice.Therefore,the use of antisense oligodeoxynucleotides（i.e.,anti-miR-33）to antagonize miR-33 may be a new strategy for the treatment of AS diseases.However,there are still considerable challenges in the clinical transformation of microRNA targeted therapy for AS.Oligonucleotides usually show poor stability,and they are easily hydrolyzed by nucleases in blood flow,target tissues and target cells,leading to inactivation before reaching target genes in cells.In addition,their nonspecific distribution after systemic administration may lead to miss target effects and serious side effects.The previous research of our group found that yeast microcapsules with content removed can be targeted to tumor and inflammation sites by oral route through bionic approach.Therefore,in this subject,through the similar principle,the oral anti-miR-33 targeted delivery system for treating AS was constructed by imitating the way of bacteria infecting the body through the intestinal tract and using yeast microcapsules as the carrier.Methods1.Preparation of blank yeast capsuleBlank yeast capsules（YC）were prepared by removing the inner contents of Baker’s yeast by culturing with acid,alkaline and organic solvent.YC were then freeze-dried.Its structure was verified by SEM and TEM,and its particle size and zeta potential were measured by DLS.2.Synthesis and Characterization of Anti-miR-33/Yeast Microcapsule Oral Delivery System（YC-ANM33）2.1 Preparation and characterization of YC-PEIThe dimethyl sulfoxide（DMSO）solution of fluorescein isothiocyanate（FITC）was mixed with the DMSO solution of polyethyleneimine（PEI）,and then the FITC labeled PEI aqueous solution（PEI-FITC）was obtained by dialysis.A certain amount of YC was ultrasoniced until it was dispersed,and then mixed with FITC-PEI aqueous solution.The solution was co-incubated,and freeze-dried to obtain YC-PEI-FITC.YC-PEI without fluorescent label was prepared by the above method.Its structure was verified by SEM,TEM and confocal microscope（CLSM）,and its particle size and surface potential was measured by DLS.2.2 Preparation and characterization of YC-ANM33Anti miR-33（ANM33）was mixed with YC-PEI aqueous solution,incubated together,centrifuged and washed,and the precipitate obtained was freeze-dried to obtain YC-ANM33.YC-ANM33-Cy3 containing Cy3 fluorescent label was alos prepared by the above method.Its structure was verified by SEM,TEM and CLSM,and its particle size and surface potential were determined by DLS.2.3 In vitro release performance evaluation of YC-ANM33YC-ANM33-Cy3 was mixed with dd water,PBS buffer and normal saline respectively,and then vibrated at 37℃to verify its hydrolysis stability in general environment.YC-ANM33-Cy3 was mixed with（1）pH 1.2 phosphate buffer solution and gastric simulant solution with pepsin and mouse gastric tissue homogenate,co-incubated for 2 h.then YC-ANM33-Cy3 was transferred to（2）pH 7.4 phosphate buffer solution and small intestinal simulant solution containing bile salt,trypsin and mouse intestinal tissue homogenate co-incubated for more than 20 h to study its release behavior under the simulated gastrointestinal environment.3.Evaluation of macrophage phagocytosis of YC-ANM33Incubate YC-ANM33-Cy3 of different doses with mouse macrophages（RAW264.7 and BMDM）for different hours,wash away the phagocytic microcapsules,and stain the nuclei and lysosomes.The phagocytosis of RAW264.7 cells and BMDM cells was evaluated qualitatively by CLSM.Flow cytometry（FCM）was used to quantitatively analyze the phagocytosis of cells.4.Effect of YC-ANM33 on the ability of macrophages to absorb and efflux cholesterolRAW264.7 cells were pretreated with YC-ANM33,washed and co incubated with NBD cholesterol.After cleaning,the cells were lysed,and the NBD fluorescence intensity was detected to verify the effect of YC-ANM33 on the cholesterol uptake ability of RAW264.7cells.The RAW264.7 cells were pretreated with NBD cholesterol and washed away,and treated with YC-ANM33.The cells were incubated with HDL in the culture medium.After cleaning,the cells were lysed,and the NBD fluorescence intensity was detected to verify the effect of YC-ANM33 on the cholesterol excretion ability of RAW264.7 cells.5.Effect of yc-anm33 on the formation of foam cellsRAW264.7 was treated with lipopolysaccharide（LPS）,stimulated/unstimulated,washed off,intervened with YC-ANM33,and then treated with oxidized low density lipoprotein（oxLDL）.After fixation,oil red O（ORO）staining was performed to observe the formation of foam cells.Using the above pretreatment method,after hematoxylin staining,100%isopropanol was used to extract ORO in cells,and the formation of foam cells was semi quantitatively detected.6.Study on the related mechanism of YC-ANM33 on cholesterol transport of macrophagesAfter the RAW264.7 cells were treated with YC-ANM33,the expression of ABCA1 and ABCG1 was detected by fluorescence quantitative pcr and Western blot.Flow cytometry was used to quantitatively detect the expression of ABCA1,and CLSM was used to observe the expression of ABCG1.7.Body distribution after oral administration of YC-ANM33After ApoE^-/-mice successfully established atherosclerosis model by high-fat diet,YC-ANM33-Cy3 with therapeutic dose was administered orally（p.o）.At 3 h and 12 h after administration,the hearts,aortas,intestines and main organs of the mice were taken for in vivo imaging.8.Aortic plaque targeting effect and oral transport pathway of YC-ANM33Atherosclerosis model ApoE^-/-mice and healthy C57 mice were orally administered with YC-ANM33-Cy3 and PEI-ANM33-Cy3 encapsulated in yeast free microcapsules.After 3 h,mice hearts,aortas and main organs were taken for in vivo imaging to investigate the targeting effect of YC-ANM33-Cy3 on plaque in the heart and blood vessels.The small intestine,Peyer collecting lymph node（PP）,mesenteric lymph node（MLN）and inguinal lymph node（ILN）of mice were taken for imaging observation in vivo to investigate the transport pathway of YC-ANM33-Cy3 after oral administration.9.Pharmacodynamic study of YC-ANM33 in treating atherosclerosisApoE^-/-mice successfully established atherosclerosis model by western diet,and then were treated with high and low dose PEI-ANM33 and YC-ANM33 respectively for 60 days.After being killed,the whole aorta was separated,and the peripheral adipose tissue of the aorta vein was removed.After longitudinal dissection,ORO staining was performed to investigate the growth of aortic plaque.The aortic root were sectioned to evaluate the plaque area,macrophage content and plaque stability of the aortic root.Western blot was used to verify the effect of YC-ANM33 on the expression of ABCA1 and ABCG1 in tissues.10.Preparation and characterization of yeast microcapsule oral delivery system（YC-AA）with ANM33 and atorvastatinA certain amount of atorvastatin powder was weighed and mixed with the DMSO solution of PEI under ultrasound,and then transfered to a dialysis bag for dialysis for 24 h,centrifuged and freeze-dried to obtain PEI-ATOR self-assembled nanoparticles.The drug loading efficiency was detected by HPLC.YC-ATOR could be obtained by co-incubation of PEI-ATOR nanoparticles with blank YC;YC-AA was obtained by co incubation of PEI-ANM33 nanoparticles with YC-ATOR.10.Pharmacodynamic study and safety evaluation of YC-AA in treating atherosclerosisApoE^-/-mice ware successfully established atherosclerosis models by western diet,and then treated with atorvastatin,YC-ATOR,YC-ANM33 and YC-AA respectively for 60 days.After being killed,the whole aorta was separated,and the peripheral adipose tissue of the aorta vein was removed.After longitudinal dissection,ORO staining was performed to investigate the growth of aortic plaque.The aortic root were sectioned to evaluate the plaque area,macrophage content and plaque stability of the aortic root.After anti-AS therapy of ApoE^-/-mice,the body weight change,organ index,blood routine analysis,liver and kidney functions,and H&E staining of main organs were recorded to investigate the safety of YC-AA in treating AS.Result1.SEM image shows that the budding hole on YC surface opened after acid-base penetration treatment,and TEM image shows the content of yeast cell successfully removed.Blank YC surface potential（Zeta potential）is-8 mV.The surface potential（Zeta potential）after PEI loading is 19 mV.After loading ANM33,the surface potential（Zeta potential）is 6mV.The encapsulation efficiency of YC to ANM33 reached 98%.The particle size of YC-ANM33 is 2-5μm.2.YC-ANM33 has good stability in neutral environment;The release is slower in the simulated stomach environment and faster in the simulated small intestine environment.3.YC-ANM33 can be phagocytosed by RAW264.7 cells in a time-dependent and concentration dependent manner.4.YC-ANM33 can significantly increase the expression of cholesterol reversal transport related proteins ABCA1 and ABCG1,thus effectively promoting the cholesterol efflux of macrophages.In addition,YC-ANM33 can effectively inhibit the transformation of macrophages into foam cells.5.YC-ANM33 was enriched in PP node of small intestinal epithelium 3 hours after oral administration,and could effectively target atherosclerotic plaque 12 hours later.6.YC-ANM33 long-term treatment can significantly increase the ABCA1 and ABCG1expression in aortic plaque tissue.It can reduce macrophage infiltration and cholesterol crystal load in aortic plaque,thus effectively reducing the necrotic core area in aortic root plaque,improving the content of plaque collagen,enhancing plaque stability,and slowing down the AS progression.7.Oral YC-ATOR has a better anti-AS effect than oral free atorvastatin.YC-AA combined treatment can significantly inhibit the occurrence and development of AS plaque and improve plaque stability,with great anti-AS effect and safety.Conclusion1.After preloading with positively charged PEI macromolecules,negatively charged ANM33 can be successfully loaded into blank YC through electrostatic interaction.2.YC-ANM33 can be successfully phagocytized by macrophages,and can increase the expression of genes and proteins related to cholesterol reverse transport in cells by silencing miR-33,promote RCT,and inhibit the transformation of macrophages into foam cells.It has a certain anti-AS effect in vitro.3.Oral administration of YC-ANM33 delivery system can target the lesion site of aorta through intestinal immune system,effectively treat atherosclerosis by promoting RCT,reducing plaque formation,and maintaining plaque stability.4.The results of in vitro and in vivo experiments show that YC-AA has good biological safety in tissues and cells.