Construction Of Dual-targeted Nanocarriers For Delivery Of Rapamycin And Their Applications In Antiatherosclerosis

Cardiovascular diseases(CVD)are kinds of global health problems cauinge the most deaths worldwide,while the morbidity and mortality are still rising.Atherosclerosis(AS)is the most important factor leading to CVD.The pathogenesis of AS is still unclear,while the inflammation hypothesis has always attracted attention among a series of hypotheses about the pathogenesis of it.Briefly,monocytes are recruited from the blood to the inflammation site of the arterial vessel wall,locally differentiate into inflammatory macrophages and foam cells.Then,the inflammatory macrophages and foam cells further promote the local inflammatory process,stimulating the development of plaque and the formation of thrombus.Therefore,inflammation can be used as a feasible strategy to combat AS lesions to achieve more effective prevention and treatment.Rapamycin(RAP),a new type of macrolide immunosuppressant,has been originally used as an antifungal drug and found an immunosuppressive effect through research.There were also many studies using it to treat arteriosclerosis.However,easily soluble in organic solvents and the slightly water-soluble nature of RAP will cause low bioavailability.Therefore,researches on a safe and effective delivery system are vital for its clinical applications of RAP.The?_v?₃ integrin overexpressed both on the surface of activated endothelial cells and macrophages accumulated in AS lesions can be an effective target for AS.A short peptide containing arginine-glycine-aspartic acid(RGD)can bind to the?_v?₃ integrin receptor and has a wide range of applications in anti-AS targeted therapy.Therefore,the study on RAP-loaded nanoparticles with RGD peptide targeting is a promising way to treat AS.In this paper,an acid-sensitive carrier called poly-1,4-diylacetone dimethyl ketone(PK3)was first synthesized independently.Polylactic acid glycolic acid(PLGA)and egg phosphatidylcholine(egg PC)were co-encapsulated with RAP to prepare composite nanoparticles,which targeted inflammation sites by their acid sensitivity.Taking the advantages of the overexpression of integrin?_v?₃ on the endothelial cell surface at the inflammation site,a cyclic peptide c RGDfc(cyclo(Arg-Gly-Asp-d-Phe-Cys))that can specifically bind to?_v?₃ was selected as the nanoparticle target.Thus,giving the nanoparticles the ability to actively target,and further explore the therapeutic effects of targeted nanoparticles.The specific content includes the following parts:1.Establishment of in vitro analysis method of RAPIn this chapter,an analytical method for in vitro detection of RAP was constructed through the detection of high performance liquid chromatography.The absorption peak of RAP was the largest at 278 nm measured by fluorescence spectrophotometer.It has been verified that the specificity of the method was good.And the accuracy was between 98%and 102%,RSD was less than 2%,which met the requirements.2.Synthesis of PK3 and preparation and characterization of dual-targeted polymer nanoparticlesIn this chapter,the ketal reaction between 1,4-cyclohexanedimethanol(CDM),1,5-pentanediol(1,5-Pe D)and 2,2-dimethoxypropane(DMP)were used to synthesize PK3.The properties of the synthesized polymer were characterized by ¹H NMR,FTIR,and X-ray diffraction(XRD).The synthesized polymer had the correct structure,met the test standards,and could be used for follow-up experiments.PK3,PLGA and egg PC were used to prepare polymer nanoparticles RAP-NPs,and c RGDfc modified dual-targeting polymer nanoparticles RAP-NPs-RGD,with particle size,polydispersity coefficient(PDI),potential(Zeta),drug loading(DL%)and encapsulation efficiency(EE%)as indicators,finally the prescription was optimized.The optimally formulated nanoparticles had particle size of 182.7±0.1nm,PDI of 0.273±0.079,Zeta of-11.83±2.76 m V.In the in vitro studies,RAP-NPs-RGD showed good stability and acid sensitivity.In the hemolysis experiment,the nanoparticles hardly hemolyze,showing good biological safety.3.Evaluation of biological effects of dual-targeted polymer nanoparticlesIn cell uptake and vitro anti-inflammatory experiments,compared with the free drug group,nanoparticles significantly increased the uptake efficiency of RAP by endothelial cells and the anti-inflammatory effect on inflammatory cells.In cytotoxicity experiments,nanocarriers are almost non-toxic to cells and have good safety.In tissue distribution experiments,compared with mice injected with RAP,nanoparticles can effectively deliver RAP to inflammatory sites of AS and prolong the circulation time of RAP in the body.In the in vivo pharmacodynamic experiment,compared with the mice injected with RAP,RAP-NPs-RGD can reduce the plaque area and significantly relieve the AS inflammation of the mice(P<0.001).In the safety experiment,the nanoparticles did not cause any damage to the organs of the mice.In summary,nanocarriers based on dual targeting of the inflammatory microenvironment are a promising delivery system for the treatment of AS.