| The development of inflammation,especially chronic inflammation,will lead to many complications and even cancer,and has adverse effects on human health and life.In recent years,the incidence rate of various inflammatory diseases such as rheumatoid arthritis and chronic pancreatitis is increasing,which is a growing threat to human beings and society.Macrophages,a very important regulator in inflammation,will be activated and infiltrated into inflammatory sites,which will further aggravate the pathological development of the disease.Therefore,activated macrophages are an ideal target for effective treatment of inflammation.Early studies in our laboratory showed that albumin nanoparticles loaded with anti-inflammatory activate ingredient celastrol(CLT)had good anti-inflammatory effect.In order to achieve more significant anti-inflammatory effect by further extending the blood circulation time in vivo,palmitic acid(PA)modified bovine serum albumin(BSA),namely PAB,was synthesized.And CLT loaded BSA nanoparticles(CLT-BSA NPs)and CLT-PAB NPs were prepared by the same solvent evaporation method.Compared with BSA,PAB was more electronegative and lipophilic,and the amount of α-helix was more,thus PAB tended to fold.After being prepared as nanoparticles,the zeta potential of CLT PAB NPs was about-30 m V,which was more negative than that of CLT BSA NPs about-20 m V.And the particle size of CLT-PAB NPs was about 100 nm,which was smaller than that of CLT-BSA NPs about 115 nm,and the polydispersity index(PDI)of CLT-PAB NPs was also smaller than that of CLT-BSA NPs.The stronger folding state and lipophilicity of PAB compared with BSA caused these results,and also made CLT-PAB NPs more integrated than CLTBSA NPs,thus PAB NPs was able to prolong CLT circulation time more effectively than BSA NPs.Surprisingly,during our experiment,PAB was found to achieve scavenger receptor-A(SR-A)targeting effect.SR-A is a receptor expressed on the surface of macrophages,and its expression level is further increased in activated macrophages.As a result of the SR-A targeting effect,PAB NPs could achieve 9.1-fold uptake of BSA NPs in inflammatory activated macrophages,and PAB could also play a specific inhibitory effect on SR-A.Furthermore,we established the adjuvant induced arthritis(AIA)rat model and caerulein induced chronic pancreatitis(CP)mouse model.In these two different animal models,the SR-A targeting effect of PAB was proved.Moreover,due to the SR-A targeting effect of PAB,PAB NPs were able to achieve significantly enhanced accumulation and longer retention time in inflammatory joints and pancreas than BSA NPs.Further experiments were carried out to explore the reason why PAB can target SR-A.The results showed that the enhanced electronegativity of PAB compared with BSA and the PA in its structure are both important for PAB targeting SR-A.Moreover,the octanoic acid with a carbon atom number of 8(the carbon atom number of PA is 16)modified BSA also has SR-A targeting effect.All those results indicate that BSA modified by fatty acids with a certain number of carbon atoms may have SR-A targeting effect.At present,the known SR-A ligands are polyanion with strong electronegativity,our research provides a new direction to design novel SR-A ligands.Activated macrophages express a variety of receptors,and CD44 is one of them.As a ligand of CD44 receptor and an important component of extracellular matrix,chondroitin sulfate(CS)has been widely used in the preparation of macrophages targeted drug delivery system due to its good properties.And there already exists a kind of SR-A targeting material,maleated BSA(MAL-BSA),whose structural foundation is also BSA.Therefore,in this study,after confirming the SR-A targeting effect of PAB,a series of in vivo and in vitro experiments have been conducted to investigate the advantages of PAB in targeted anti-inflammatory therapy compared with CS through CD44-mediated pathway and MAL-BSA with SR-A targeting.The results showed that SR-A-mediated activated macrophages targeting effect of PAB was better than CD44-mediated activated macrophages targeting effect of CS;and MAL-BSA strongly targeted SR-A but was lethal for experimental animals,thus limiting its in vivo application.Then the therapeutic effect of CLT-PAB NPs was studied in AIA rats and cerulein induced CP mice.The results showed that,in AIA rats,CLT-PAB NPs could significantly improve rheumatoid arthritis symptoms at a lower CLT dose compared with free CLT and CLT-BSA NPs because of the SR-A targeting effect and the prolonged circulation time;Compared with CLT-BSA NPs and free CLT,CLT-PAB NPs also significantly improved the status of pancreatic lesions in cerulein induced CP mice.The side effects of CLT-PAB NPs were also investigated in vitro and in vivo,the results showed that the safety of CLT-PAB NPs is better than CLT and CLT BSA NPs.The anti-inflammatory potential of PAB at organelle level was further investigated.The results showed that in activated macrophages,PAB could specifically accumulate in endoplasmic reticulum(ER)through the pathway of fatty acid uptake by ER.Octanoic acid modified BSA could also target ER,which indicated that fatty acid with a certain number of carbon atoms modified BSA might target ER through the same pathway.As the largest organelle,ER controls many physiological processes in eukaryotic cells including lipid and carbohydrate metabolism,protein synthesis,folding and transportation and so on.These results indicate that PAB with SR-A and ER double targeting effect may have great application potential in the treatment of diseases in the future.In general,PAB was synthesized in order to obtain a preparation with longer circulation time on the basis of albumin nanoparticles.However,we were surprised to discover the SR-A targeting effect of PAB and its ER targeting effect,and further proved the great potential of PAB as a new activated macrophage targeting vector in targeted anti-inflammatory therapy;and our study provides different ideas for designing new SR-A and ER targeting carrier. |
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