乳铁蛋白结合聚乙二醇聚乳酸纳米粒脑内递药特性的体内外研究胡凯莉，李靖炜，沈烨虹,陆伟，高小玲,蒋新国*（复旦大学药学院药剂教研室，上海）摘要：目的构建一种新型可生物降解脑内递药系统-乳铁蛋白结合聚乙二醇聚乳酸纳米粒（Lf-NP），并对其脑内递药特性进行体内外评价。方法采用马来酰亚胺基聚乙二醇-聚乳酸和甲氧基聚乙二醇-聚乳酸通过复乳溶剂挥发法制备纳米粒（NP）,将其与巯基化的乳铁蛋白（Lf）共价连接制得Lf-NP。采用透射电镜技术，免疫电镜技术，动态光散射分析，X-射线光电子能谱分析及酶联免疫吸附试验对Lf-NP的形态,粒径和表面Lf的连接进行鉴定和表征。以香豆素-6为荧光探针定性及定量的研究了小鼠脑毛细血管内皮细胞bEnd.3对Lf-NP的摄取情况。采用CCK-8评价了Lf-NP对细胞活力的影响。利用荧光显微技术和冷冻切片技术观察小鼠尾静脉注射载香豆素-6的Lf-NP后的脑内分布情况并进行了药动学研究。结果制得的Lf-NP形态圆整，粒径在110nm左右。免疫电镜技术，X-射线光电子能谱分析及酶联免疫吸附试验证实了Lf-NP表面共价连接有Lf，且每个Lf-NP表面的Lf数约为55。bEnd.3对Lf-NP的摄取在37℃，不同浓度和不同孵育时间条件下均显著高于未结合*基金项目：国家973项目“导向性纳米载药系统及其脑部疾病治疗与诊断中的应用基础研究”作者简介：胡凯莉，女，博士研究生?通讯作者：蒋新国，男，研究员，博士生导师Tel:(021)E-mail:xgjiang@shmu.edu.cn纳米粒（NP）。静脉注射载香豆素-6的纳米粒后，Lf-NP组小鼠脑内香豆素-6的量约为NP组的3倍。3mg/ml的Lf-NP对bEnd.3细胞的活力无显著影响。结论Lf-NP能显著增加药物的脑内递送且具有较好的生物相容性，有望作为一种新型的脑内递送系统输送蛋白多肽和基因等大分子药物入脑。关键词：乳铁蛋白；血脑屏障；香豆素-6；生物可降解纳米粒；脑内递送中图分类号：R9Lactoferrin-conjugatedPEG–PLAnanoparticleswithimprovedbraindelivery:invitroandinvivoevaluationsHUKai-li,LIJing-wei,SHENYe-hong,LUWei,GAOXiao-ling,JiangXin-guo(DepartmentofPharmaceutics,SchoolofPharmacy,FudanUniversity,Shanghai)ABSTRACT：OBJECTIVEToconstructthelactoferrin(Lf)conjugatedpoly(ethyleneglycol)–poly(lactide)(PEG–PLA)nanoparticle(Lf–NP)asanovelbiodegradablebraindrugdeliverysystemandevaluateitsinvitroandinvivodeliveryproperties.METHODSLfwasthiolatedandconjugatedtothedistalmaleimidefunctionssurroundingonthepegylatednanoparticlespreparedbydoubleemulsionandsolventevaporationproceduretoformtheLf–NP.Dynamiclightscatteringanalysis,transmissionelectronmicrograph(TEM),X-rayphotoelectronspectrometryanalysisandELISAwereusedtocharacterizetheLf-NP.Afluorescentprobe,coumarin-6wasincorporatedintoLf–NPanditsuptakebyamousebraincapillaryendothelialcellline,bEnd.3werecomparedtothatofunconjugatednanoparticle(NP).ThebraindistributionofLf-NPfollowinganintravenousadministrationwasevaluatedquantitativelybypharmacokineticstudiesandqualitativelybyfluorescentmicroscopy.TheinfluenceofLf-NPoncellviabilitywasalsoevaluated.RESULTSDynamiclightscatteringanalysisandtransmissionelectronmicrograph(TEM)showedthattheLf–NPhadaroundandregularshapewithanaveragediameterofaround110nm.TheexistenceofLfonthesurfaceofLf–NPwasverifiedbyTEMobservationandX-rayphotoelectronspectrometryanalysis.TheLfELISAresultsconfirmedthebiorecognitiveactivityofLfafterthecovalentcouplingprocedureandsuggestedtheaveragenumberofLfconjugatedoneachnanoparticlewasaround55.TheuptakeofLf–NPbybEnd.3wasshownsignificantlyhigherthanthatofNPat37℃,atdifferentconcentrationsandincubationtimes.Followinganintravenousadministrationofadoseof60mg/kginmicecaudalvein,anear3foldsofcoumarin-6werefoundinthemicebraincarriedbyLf–NPcomparedtothatcarriedbyNP.Cellviabilityexperim...