[生物医药论文精品a]苗口服接种新载体：荆豆凝集素化壳聚糖纳米粒传输系统的研究

1苗口服接种新载体荆豆凝集素化壳聚糖纳米粒传输系统的研究李凤前，费轶博，刘继勇，朱全刚，胡晋红第二军医大学长海医院药学部，上海200433摘要本文依据肠黏膜及派伊尔集合淋巴结（PEYERSPATCHES,PP）的免疫学特点，以乙型肝炎疫苗为模型药物，将能同肠道微皱褶细胞上岩藻糖特异性结合的荆豆凝集素，对壳聚糖进行修饰，构建并研究PP定位的疫苗微粒口服传输系统。离子交联高压匀化工艺制备的壳聚糖纳米粒形态较规则。用激光G6967G4568粒G5242G1214G8991定纳米粒G5191G3355粒G5464为3668NM，G3822G2010G6967系G6980为0122，粒G5242G2010G5079较G12376。以G1314G9213高G17907离G5527G8873G8991G5483乙肝疫苗纳米粒G5191G3355G2265G4565G10587G332490以上，G17902G17819SDSPAGEG11017G8903G15932G7138，乙肝疫苗G3324G2465G5224前G2530G8821G7389G2469G10995G2010子G18339的G2476化。用G6110二G18287G8975化G8873制备凝集素修饰的壳聚糖纳米粒，结合G10587G2499G1781085332。岩藻糖的G4396G3324G1262G16765BSMG994凝集素化纳米粒的结合G2475G2052G6245制，凝集素G3324结合G2052纳米粒G15932G19766G2530糖结合G8975性的G1457G6357。G14651光G7186微G19248G16278G4531G2469G10628纳米粒G3324淋巴结G1025G7389G980定G4512集G6940G7536。G994G5078G2818制G2070G13920G8892G13464G451口服乙肝疫苗G7422凝集素化纳米粒G13464G451口服乙肝疫苗G13464G451G19464性对G10043G13464（G10995G10714G11428G8712）G8616较，口服凝集素化乙肝疫苗纳米粒G13464G3324BALB/CG4579G21748，能G3324G980定G12255G5242上G5353G2469免疫G5224G12584。本研究G15932G7138，凝集素化壳聚糖纳米粒G2499以G1328为疫苗G12879药物G7389G6940的G17745G1319，用G1122肠PP的定G2533G13485药，为疫苗口服免疫G5332G17779G1114G7044G5617G17347。关键词离子交联高压匀化G8873G727G3822聚G11979G18252G19060G727凝集素化壳聚糖纳米粒G727岩藻糖G727G10287G20064G991G14158G12908G15519G11345G727疫苗口服G6521G12193G727派伊尔集合淋巴结NEWPARTICULARCARRIEROFORALVACCINATIONULEXEUROPAEUSAGGLUTININANCHOREDCHITOSANNANOPARTICLESDELIVERYSYSTEMFENGQIANLI,YIBOFEI,JIYONGLIU,QUANGANGZHU,JINHONGHUDEPARTMENTOFPHARMACEUTICALSCIENCES,CHANGHAIHOSPITAL,SECONDMILITARYMEDICALUNIVERSITY,SHANGHAI,200433,CHINAABSTRACTTHISPAPERISPROPOSEDUPONTHEIMMUNOLOGICCHARACTERISTICSOFENTERICMUCOSAANDA1A0A2A3A5A6A4A7A8A9A10A1A0A11A12A2A3A1430500639A15A13A17A16A18A9A19A20A21A22A2A23A24A140552NM040A15A26A25A27A28TEL/FAX862125070668,EMAILHUJHSMMUEDUCN2PEYERSPATCHESPPBIODEGRADABLEMATERIALOFCHITOSANWASMODIFIEDWITHTHELECTINOFULEXEUROPAEUSAGGLUTININUEA,WHICHCOULDBESPECIFICTOTHERHODEOSEWITHINTHEMICROFOLDCELLUNDERINTESTINALTRACT,ANDTHENUSEDFORNANOENCAPSULATIONTHEMODELHEPATITISBVACCINEWASTHENENTRAPPEDWITHINTHELECTINANCHOREDNANOPARTICLESFORORALDELIVERYTHEUNIFORMSHAPEDCHITOSANNANOPARTICLESWEREPREPAREDBYINONOTROPICGELATIONHOMOGENIZATIONPROCESSWITHTRIPOLYPHOSPHATEASGELATINIZERTHEMEANDIAMETEROFTHECHITOSANNANOPARTICLESWAS3668NMWITHTHEPOLYDISPERSITYINDEXOF0122THEVACCINEENTRAPMENTEFFICIENCYOFNANOPARTICLESWHICHISSEPARATEDBYCENTRIFUGALIZATIONWASABOVE90THELECTINANCHOREDCHITOSANNANOPARTICLESWEREPREPAREDWITHTHEGLUTARALDEHYDEACTIVATIONMETHODANDTHEBINDINGRATEOF85332COULDBEACHIEVEDTHEINTERACTIONSBETWEENLECTINANCHOREDNANOPARTICLESANDBOVINESUBMAXILLARYGLANDMUCINDECREASEDSTRONGLYWHENTHECOMPETINGSUGAROFLFUCOSEADDEDTHESERESULTSCLEARLYSUGGESTEDTHATTHEREMAINEDACTIVITYOFLECTINANDITSSPECIFICBINDINGCHARACTERISTICSTOFUCOSERESULTOFFLUORESCENCEMICROSCOPESTUDYSHOWEDTHATTHELECTINANCHOREDNANOPARTICLESCOULDBEENRICHEDINTHEPAYERSPATCHTHEHBSAGANTIBODYCONCENTRATIONSOFBLOODSERUMSAMPLESOFMICEINDIFFERENTTIMEWEREDETECTEDAFTERINTRAGASTRICADMINISTRATIONOFHBSAG,PLAINHBSAGNANOPARTICLES,LECTINANCHOREDHBSAGNANOPARTICLESANDPHYSIOLOGICALSALINEGROUPTHERESULTSALSOSHOWEDTHATTHELECTINANCHOREDNANOPARTICLESWEREMOREEFFICIENTTHANOTHERGROUPSEXCEPTTHEINTRAMUSCULARINJECTIONOFCLINICALDOSAGEFORMTHEABOVERESULTSSHOWEDTHATLECTINANCHOREDCHITOSANNANOPARTICLESWOULDBEAPROMISINGCARRIERFORPEYERSPATCHLOCATEDDRUGDELIVERY,ANDMIGHTREFERREDNOVELCONSIDERATIONFORTHEDEVELOPMENTOFORALDELIVEREDVACCINEDOSAGEFORMSKEYWORDSINONOTROPICGELATIONHOMOGENIZATIONPROCESSTRIPOLYPHOSPHATELECTINANCHOREDCHITOSANNANOPARTICLESFUCOSEBOVINESUBMAXILLARYGLANDMUCINORALVACCINATIONPEYERSPATCHES疫苗G3324G8981行G11161G19462G8847G7053G19766G2469G6393G18337G16213G1328用，口服G6521G12193疫苗对G1122G18337大G8981行性G11154G11161G451G12373G2469G13051G5625疫G5785的G19462G6523G5859G1053G18337大，口服疫苗G13485药系统（ORALLYADMINISTEREDVACCINEDELIVERYSYSTEM,OAVDS）能G7053G1427G1000直G6521刺激易感染部位产G10995抗G1319，G3324减少G6521G12193次G6980G451降G1314G6521G12193脱漏G10587G451提高疫苗贮运管G10714G6940G10587及简化G6521G12193G7053式等G7053G19766具G7389G18337G16213G5859G1053。因此，以胃肠道黏膜为输送部位的OAVDS，是目前疫苗G13485药系统研究最G8975跃的领域之G980。3肠系淋巴G13464织G1025G4512含的派伊尔集合淋巴结（PEYERSPATCHES,PP）对抗原的摄取具G7389特异性2，PP参G994免疫调节G1328用，是疫苗口服吸收和免疫抗G1319G2465G5224的G18337G16213部位。但口服疫苗将直G6521G2475G2052胃肠道G1025PH环境及各G12193酶系的影响，G3324抗原经M细胞G2052G17810集合淋巴G4579结PP的途G1025易被降解，其吸收G6940G10587也G1314，G3324目标G1328用部位的抗原不足，G2052G17810PP附近的疫苗G980般难以G5353G2469G7389G6940的免疫G5224G12584G2465G5224。因此，OAVDS的设计需充G2010考虑胃肠道吸收和转运的形态学屏障（上皮细胞G451黏膜）和G10995G10714学屏障（胃肠酶系G451PHG451传递G1319）1,2。微囊化（MICROENCAPSULATION）技术G3324G1457护疫苗和促进吸收G7053G19766能起G2052较好的协同G1328用，微粒系统G2499“克服”胃肠道屏障，由胃肠道M细胞以跨膜转运或细胞旁G17347的G7053式摄入。G3324微粒G15932G19766结合能识别M细胞的特异性基团或G2475G1319，G2499使疫苗的递送具G7389G980定的靶G2533性，G7389利G1122高G6940疫苗传输系统的定位设计。荆豆等外源性凝集素G2499特异性地识别M细胞膜G15932G19766上糖G15519G11345G1025G4512含的岩藻糖，并G994之结合3,4，这为口服疫苗定位滞留释放系统的研究提供G1114设计G5617G17347。考虑G2052天然高G2010子材料壳聚糖所带正G11017荷对肠上皮G13051密结合点的G5332放机制5，能促进肠道上皮细胞对大G2010子物质的吸收6，G1000壳聚糖具G7389对G15519G11345G7389利的结合位点。本文以其为G17745G1319材料，G5224用离子交联高压匀化工艺制备壳聚糖纳米粒，采用微G18339G6110二G18287G8873对壳聚糖纳米粒进行凝集素化修饰，验证G1114荆豆凝集素化纳米粒的糖结合特异性，进G980步考G4531G1114凝集素化纳米粒G3324PPG1025的定位G5785况及其G3324G4579G21748G1319内抗G1319滴G5242G2476化G5785况。该研究工G1328，G2499对凝集素化微G17745G1319G1328为疫苗经口服胃肠道G6521G12193的G2499行性提供实验依据和技术借鉴。1仪器、材料、试剂及动物DF101S集G9921式G5670G9213G2164G9921G11925G2159G6617G6304G3132（G5053G1053G5078G14533G4798G1116G2338G1214G3132G2390制G17908）G727EMS2型G2164G9921定G7114G11925G2159G6617G6304G3132（天G8953G8443G16846G1214G3132G1214G15932G7389G19492G1856G2508）G727高压G3355质机AH110D（ATSENGINEERINGNCORPORATED）G727全G14270G2172酶标G2010G7524G1214ELX800（G13666G3281BIOTEKG1856G2508）G727ZLS型G11017位/粒G5242激光G6967G4568G8991定G1214BARBARA,USAG727G2500式高G17907G1931G1935离G5527机（SUPERT21,SORVALL,USA）G727G17891G4568G11017子G7186微G19248（HITACHIH600，G7097本G7097G12447G1856G2508）。10AG4719G8953HPLC系统G727凝G14026G14406G16901G7621（TOSHOG727TSKGELG2000SWXL78300MM）。乙肝疫苗（G2283G1152天G3375G10995物制G2709G1856G2508，G2010子G1833924KDA）G727壳聚糖（上海G3926G2525G10995物G12197技G2469G4649G7389G19492G1856G2508，脱乙G18244G524290G705，G5191G3355G2010子为4080KDA）G727G989聚G11979G18252G19060（G3281药集团化学G16809G2070G7389G19492G1856G2508，化学G13443）G727G1924G18271G18252（G3281药集团化学G16809G2070G7389G19492G1856G2508）G2010G7524G13443G727MICROBCAG16809G2070G11430（G13666G3281PIERCEG1856G2508）G727荆豆凝集素，BSM，岩藻糖（SIGMA）。48G2620G21579BALB/CG4579G21748（202G），由本G7669G2172物实验G1025G5527供G5224，合G7696证G2507G726SCXKG883020030006。2方法21G256离子交联匀化G8873G257制备壳聚糖纳米粒7将CSG9354G1122G12244G18271G18252G8712G9354G9094G1025（G19560G3824G9354G13972），G18209G611602（W/V）的壳聚糖G9354G9094，TPPG9354G1122G14988G20323G8712G18209G611602（W/V）的G9354G9094。G3324不G7041G6617G6304G991（G11925G2159G6617G6304，600RPM），将TPPG9354G9094G13543G5942滴入壳聚糖G9354G9094G1025（滴G17907G13434为3ML/MIN），由G9560G7138G9354G9094G17892G9188转G2476为G2588G10628G9141G15025G14406G1095光的G14026G1319G9354G9094G1319系，G7693据G1095光G5390G5381G2499G2040G7041纳米粒的形G6116。将离子交联制G5483的壳聚糖G14026G1319G9354G9094G1319系进G980步经高压匀化技术G3800G10714。调节G1095匀机的压G2159，G980G13435G19412压G2159600BAR，二G13435G19412压G215960BAR，将用离子交联G8873制备的G14026G1319G9094G13634入高压G1095匀机G1025，G1095匀G2530收集G7691G2709，G1889G13634入G1095匀机，G2465G3809G3247次，收集G7691G2709，G5483G2052纳米粒。22壳聚糖纳米粒的外G16278形态及粒G54648取纳米粒G14026G1319G9163G5760G90941G7942滴G13634G1122G19120G13605上，用2G705的G11979G19068G18252进行G17139染，以G17891G4568G11017子G7186微G19248G16278G4531纳米粒的形态并G6305G10043。取少G18339的CSNPSG9163G5760G9094，用G14988G20323G8712G12244释3G1505，用激光粒G5242G2010G7524G1214G8991定纳米粒的G5191G3355粒G5464。G8991定G7477G1226G726光源G726HENE激光（06328）G727G1183质G726G8712G727G11017G3342G5390G5242G726EG72910V/CMG727粒G5464G6967G4568G16294G72690A29，G8611G13464G18337G3809实验G989次。23荆豆凝集素对壳聚糖纳米粒的结合及修饰将以“离子交联G8873匀化工艺”制备的壳聚糖纳米粒G9163G5760G9094进行离G5527，离G5527G7477G1226G72620000RPMG45130MIN。G5335G2447上G9177G9094，G991G4630纳米粒用PBSG8939G90803次，G8939G2447G7422参G994交联G2465G5224形G6116纳米粒的壳聚糖。然G2530G2533G2010离G5483G2052的G991G4630纳米粒G1025，G2164入少G18339PBSG9354G9094，G9469G9077使之G3355匀G2010G6967。G2164入G17878G18339G6110二G18287G9094（25，W/V），将G9163合G7691G2709G13634G1122G5670G9213G8712G9032G6683G5214上G6403G66836G4579G7114（G9213G5242G72625G263，80G6403次/G2010G19059）。G6403G6683G8975化的壳聚糖上G8975性伯胺基，以PBSG8939G90803次，以除G2447G7422G2465G5224的G6110二G18287G9354G9094，将离G5527G2530的沉淀物G2010G6967G1122PBSG9354G9094G1025，备用。G2533经G6110二G18287G8975化的壳聚糖纳米粒G1025G2164入荆豆凝集素G9354G9094，常G9213放G13634G17819G3824。离G5527G2010离上G9177G9094。采用所建G12447的G15519G11345凝G14026G14406G16901G2010G7524G8873G8991定凝集素的含G18339，G7693据G8991G5483的游离凝集素G18339和G2164入总G18339的差值，计算凝集素G3324壳聚糖纳米粒上的结合G10587，进G980步考G4531胺基G8975化G2070G6110二5G18287对凝集素结合G10587的影响。24凝集素化纳米粒G994G10287G20064G991G14158G12908G15519G11345（BOVINESUBMAXILLARYGLANDMUCIN,BSM）的结合G16809验BSM是凝集素特异性结合G2475G1319，但当凝集素G8975性G2469G10995改G2476G7114，BSM即不能G994凝集素G7389G6940结合，利用该性质G2499以检验凝集素G8975性。将凝集素化壳聚糖纳米粒G9163G5760G9094同BSM的PBSG9094G9163合，G9469G9077G9163G57605SEC，室G9213孵育3H，G2530离G5527（5000RPM，15MIN）G2010离G7422结合的BSM，G8991定上G9177G9094G1025的BSMG18339，G7693据G8991定值和G2164入总G18339的差值，计算凝集素化壳聚糖纳米粒对BSM的结合G10587。25岩藻糖对凝集素化纳米粒同BSM结合的竞争G6245制G1328用岩藻糖是G8975性凝集素特异性结合G2475G1319，G2499以竞争BSMG994凝集素化纳米粒的结合G1328用，降G1314BSM对凝集素化纳米粒的结合。G3926G7536凝集素的G8975性丧失，则不能G1319G10628出竞争G6245制结合G10628象，由此将进G980步考G4531凝集素化纳米粒上荆豆凝集素的G8975性。同上G8873，G3324纳米粒和BSM的结合G17819G12255G1025G2164入岩藻糖，G8991定结合G10587G2476化G5785况。26疫苗的G2265G17745及G2265G4565G10587采用G256两步G8873G257，将制备好的凝集素化壳聚糖纳米粒和乙肝疫苗G9354G9094G9163合，G9213孵2H。然G2530取1ML乙肝疫苗纳米粒，G13634入离G5527机G1025离G5527（20000RPM，30MIN，10），取上G9177G9094，用凝G14026G14406G16901G7621G8991定上G9177G9094G1025乙肝疫苗含G18339，按G991式计算壳聚糖纳米粒的G2265G4565G10587和G17745药G18339。G2265G4565G10587G729投入量上清投入量WWW100G705G727G17745药G18339（G/ML）G729材料总量上清投入量WWW27凝集素化纳米粒G3324肠PPG1025的定位将G14651光素G2164入凝集素化纳米粒，壳聚糖纳米粒G1025，G112237孵育3H，取出，制备G14651光素化纳米粒。BALB/CG4579G21748禁食G980天，G9177G10714肠胃。口服凝集素化纳米粒，G989G4579G7114G2530解剖G2530G12447即G2010离G4579肠，用37抗G10995素G9094G8939G9080。G8611只G2172物取出6段（G8611段3CM）G4579肠，前3段从空肠（无派伊尔淋巴结）取G5483，G25303段从回肠（G7389派伊尔淋巴结）取G5483，用37G10995G10714G11428G8712灌G8939G989次。G1457G4396G1122甲G18287G9354G9094G1025，G1931G1935切片，用G14651光G7186微G19248G16278G4531切片，G8616较凝集素化纳米粒和非6凝集素化纳米粒对G1122派伊尔淋巴结靶G2533性的G8616较。28疫苗纳米粒经G4579G21748灌服G2530的血G9177抗G1319滴G524225只BALB/CG4579G21748随机G2010为五G13464，AG726G13920内G8892G4568G13464（G5078G2818乙肝疫苗G16809G2070），BG726口服凝集素化乙肝疫苗纳米粒G13464（G14270制制G2070），CG726口服G7422凝集素化乙肝疫苗纳米粒G13464，DG726口服乙肝疫苗，EG726口服G10995G10714G11428G8712（G19464性对G10043G13464）。G13485药前2G4579G7114，G4579G21748禁食，灌胃100L75的碳G18252氢G19060G9354G9094以G1025和胃G18252。G8611G13464G4579G21748按G10043G13464别G2010别G13485药。AG726G4579G21748肱G13920G8892G456801MLG5078G2818乙肝疫苗制G207010G/MLG727BG13464G726G13464灌胃05ML凝集素化纳米粒制G2070，G2265G17745乙肝疫苗01ML100G/MLG727CG13464G726灌胃05MLG7422凝集素化纳米粒制G2070，G2265G17745乙肝疫苗含G1833901ML100G/MLG727DG13464G726灌胃乙肝疫苗（20G/ML05MLG727EG13464G726灌胃G10995G10714G11428G871205ML。G13485药两G2620G2530，眼眦静脉丛取血，G8611只G4579G21748取出400L左右血G9094，离G5527（10000RPM，30MIN），G2010离上G9177，G1935G112270，待G8991。同G7114按G10043前次G2010G13464进行G2164G5390免疫，G2010别G11224G4516G4518G45112G45124G2620眼眦静脉取血，G2010离血G9177，G1935G112270，待G8991，实验结束G7114用乙肝G15932G19766抗G1319检G8991G16809G2070G11430G8991定。3结果与讨论31壳聚糖纳米粒及相关性质壳聚糖是G14270然界来源第二大丰G4512的亲G8712性G3822糖，具G7389G10995物G12908附性和G10995物相容性，广泛G1328为药用辅料。1989年，BODMEIER等首次报道G1114离子交联G8873用G1122制备壳聚糖纳米粒的制备，利用壳聚糖的游离氨基G994TPPG19464离子G2469G10995G2010子间或G2010子内交联G2465G5224，从而制备壳聚糖珠球状凝G14026。由G1122该实验G2465G5224G7477G1226G9213和，易G1122调G6523G2465G5224结G7536，不使用G7389机G9354G2070，特别G17878G1122G3822肽G451G15519G11345质等G980G12879大G2010子药物的G2265G17745。离子交联G8873是利用CS的正G11017性G994TPP的G17139G11017性产G10995的静G11017G5353G2159G1328用而形G6116G2010子内或G2010子间交联G2465G5224，从而G14270G2469形G6116纳米G13435微粒。7FIG1TEMPHOTOGRAPHSOFCHITOSANNAMOPARTICLESBYINONOTROPICGELATIONLEFTANDBYINONOTROPICGELATIONHOMOGENIZATIONPROCESSRIGHT图1A为离子交联G8873制备的CSNPS的G17891G4568G11017G19248G10043片，G2499见，纳米粒形态不规则，部G2010G2588G13605状结构。图1B为经G17819高压匀化的壳聚糖纳米粒图片，G2499见，经G17819匀化G2530，纳米粒外G16278规则较圆整，大G4579较G3355匀。图14为G8821经匀化的纳米粒的粒G5464G2010G5079图，纳米粒粒G5464G2010G5079较宽，图2为经G17819匀化前G2530纳米粒粒G5464G2010G5079图，G2499以看出纳米粒粒G5464G2010G5079G7138G7186G2476G12376。FIG2THESIZEDISTRIBUTIONSOFCROSSLINKEDCHITOSANNANOPARTICLESBEFORELEFTANDAFTERHOMOGENIZATIONRIGHT离子交联G8873是利用荷正G11017的CSG994荷G17139G11017的TPPG2010子之间的静G11017G5353G2159G1328用而形G6116的，但是由图2G2499以看出，形G6116的纳米粒的粒G5464G3822G2010G6967系G6980较大，即纳米粒粒G5464较不G3355匀，长G7114间放G13634就G1262形G6116沉降G10628象，影响纳米粒制G2070的稳定性。微粒G1319系G1025，微粒粒G5464G3355匀G5242是G1319系是否稳定的G980个G18337G16213评价指标，粒G5464不G3355匀，粒子容易沉降，稳定性差。8高压G1095匀是G3324近室G9213G7477G1226G991，G17902G17819调节高压G1095匀机的压G2159，使高压产G10995的G5390大推G2172G2159使纳米粒G17902G17819G1095匀机的细孔，从而匀化纳米粒的粒G5464。G1095匀前G2530，纳米粒粒G5464的G3822G2010G6967系G6980由04左右降G1314G205201左右，这G2499以说G7138，G17902G17819高压G1095匀，纳米粒的粒G5464G2476G5483更G2164G3355匀，从而提高纳米粒的稳定性。32G6110二G18287G2164入G18339对凝集素G994壳聚糖纳米粒结合G10587的影响凝集素G994微G17745G1319G15932G19766官能团G2469G10995共价结合，结合主G16213G2010为G8975化和连G6521两个G17819G12255。G8975化G17819G12255是修饰的关键，主G16213将微G17745G1319G15932G19766的官能团转化为能够G994凝集素G15932G19766氨基结合的基团，或者将特殊基团G5353入凝集素G2010子，G17902G17819该基团G994微G17745G1319官能团特异结合，而将凝集素结合G2052微G17745G1319，主G16213G7389碳化二亚胺G88739G451G6110二G18287G887310G451硫醇化G887311等。而碳化二亚胺G8873主G16213针对G1122G15932G19766G4396G3324羧基的微粒。硫醇化G8873G3324G4396G3324G7389顺丁烯二G18244亚胺的PEGG7114才G2499G2469G10995。本文G5224用的G17745G1319材料壳聚糖，G15932G19766G4396G3324大G18339的游离氨基，故选择G6110二G18287G8975化G8873来实G10628荆豆凝集素对壳聚糖纳米粒的修饰。CHOCH23CHOGLUTARALDEHYDEPH74CHOCH23CHCCH22NCHOCHONH2CHITOSANCHOCH23CHCCH22NCHOCHNCHITOSANUEANNUEAUEANH2CHCH23CHCCH22NCHCHNCHITOSANFIG3CHEMICALREACTIONSINVOLVEDDURINGTHEACTIVATIONOFCSWITHGLUTARALDEHYDEANDBOUNDOFUEAONTOTHEACTIVATEDCSG3926图3所示，G6110二G18287首先聚合G6116为G3822G18287，G3822G18287G994壳聚糖G15932G19766的游离氨基G17902G17819氨G18287缩合G2465G5224结合，从而将G18287基G5353入壳聚糖G15932G19766，至此称之为G8975化G17819G12255。G6521着，凝集素G15932G19766的氨基G994携带G18287基的壳聚糖G2010子G2469G10995氨G18287缩合，从而将凝集素连G6521G2052壳聚糖G2010子10。90204060801000100200300400500AMOUNTOF25GLUTARALDEHYDELLECTINBOUNDEDFIG4EFFECTOFINCREASINGCONCENTRATIONINTHEBINDINGOFULEXEUROPAEUSLECTINTOCHITOSANNANOPARTICLESEXPERIMENTALCONDITIONSROOMTEMPERATURE,REACTIONTIME6H,PBSPH74,N3由图4G2499见，随着G6110二G18287G2164入G18339的增G2164，凝集素G994壳聚糖纳米粒的结合G10587G17892G9188增G2164，当G6110二G18287G2164入G18339为200LG7114，结合G10587G17810G2052最大（80G705），当G6110二G18287的G2164入G18339增G2164G2052500LG7114，结合G10587G2465而G991降。这G2499能由G1122增G2164G8975化G2070G6110二G18287的G18339G7114，G5353入壳聚糖G15932G19766的G18287基增G2164，则相G5224的G994凝集素结合G10587就增G2164，然而，壳聚糖G2010子G15932G19766的结合位点也是G7389G19492的，当G2164入G17819G3822G18339的G6110二G18287G7114，并不能使G18287基都结合G2052壳聚糖G2010子的G15932G19766，同G7114由G1122壳聚糖G2010子G15932G19766的G18287基G994凝集素的氨基的结合也G4396G3324G980定饱和性的，所以G2588G10628这G7691的结合趋势。33凝集素化纳米粒G994G10287G20064G991G14158G12908G15519G11345（BOVINESUBMAXILLARYGLANDMUCIN,BSM）的结合G16809验BSM是凝集素特异性结合G2475G1319，但当凝集素G8975性G2469G10995改G2476G7114，BSM即不能G994凝集素G7389G6940结合，利用该性质G2499以检验凝集素G8975性。100204060801000100200300400500AOUNTOF1MG/MLBSMLINTERACTEDMUCINCSNPUECSNPFIG5KINETICSOFBSMINTERACTIONWITHULEXEUROPAEUSLECTINANCHOREDCHITOSANNANOPARTICLESUECSNPANDCHITOSANNANOPARTICLESCSNPUSEDASCONTROLSEXPERIMENTALCONDITIONSROOMTEMPERATURE,PBSPH74,N3图5G7186示G726BSMG2499以同壳聚糖纳米粒结合，随着BSM用G18339的增G2164，结合G10587G2465而G991降，说G7138BSM同壳聚糖结合具G7389饱和G10628象。凝集素化壳聚糖纳米粒同BSM的结合G10587，G7138G7186高G1122G7422凝集素化的壳聚糖纳米粒，G1319G10628G1114荆豆凝集素的G8975性。当BSM的G2164入G18339为200LG7114，BSM同凝集素化纳米粒的结合G10587G994空G11345纳米粒的结合G10587间差值最大。34岩藻糖对凝集素化纳米粒同BSM结合的竞争G6245制G1328用岩藻糖是G8975性凝集素特异性结合G2475G1319，G2499以竞争BSMG994凝集素化纳米粒的结合G1328用，降G1314BSM对凝集素化纳米粒的结合。G3926G7536凝集素的G8975性丧失，则不能G1319G10628出竞争G6245制结合G10628象，由此将进G980步考G4531凝集素化纳米粒上荆豆凝集素的G8975性。FIG6BSMBINDINGTOUECSNPANDCSNPINSUSPENSIONSWITHOUTAORWITHB100MMOLFUCOSEEXPERIMENTALCONDITIONSROOMTEMPERATURE,INCUBATIONTIME1H,PBSPH74,50556065707580859095100UENPNPUENPNPINTERACTEDMUCINAB11N3图6为岩藻糖G2164入前G2530，BSM结合G10587的G2476化G5785况。G2499以看出G726荆豆凝集素化壳聚糖同BSM的结合G10587G7186著G991降，由87G705降至74G705。而G7422凝集素化的壳聚糖纳米粒同BSM的结合G10587几乎G7422G2476化，G13512G6357G332470G705左右。G15932G7138荆豆凝集素同BSM的结合G1328用，G2499以被岩藻糖竞争G6245制，G2465G5224出凝集素化壳聚糖纳米粒上G19182合的凝集素G8975性G1457G6357。岩藻糖是除BSM外的G2490G980特异性结合G2475G1319。将G980定G18339的岩藻糖G2164入BSMG9354G9094G1025，岩藻糖G994凝集素的特异性结合G5529定G6245制BSMG994凝集素化纳米粒的结合，所以G2164入岩藻糖的BSMG9354G9094G994凝集素化纳米粒的结合G10587G8616BSMG9354G9094凝集素化纳米粒结合G10587G1314，从而验证G1114凝集素G3324连G6521G2052壳聚糖纳米粒G15932G19766G2530G8975性的G1457G6357G5785况。35G451乙肝疫苗的G2265G17745及其G3324纳米粒G1025的G2265G4565G10587和G17745药G18339G8991定G2265G4565G10587和G17745药G18339是评价纳米粒制G2070质G18339和制备工艺的G18337G16213指标，因此本研究进行G1114G2265G4565G10587和G17745药G18339G8991定G7053G8873的考G4531。G8991定纳米粒G2265G4565G10587及G17745药G18339的G7053G8873主G16213G7389G17241G9400G8873G451G4630G7524离G5527G8873（即G12907G2709经SEPHADEXG50G14901聚糖凝G14026G7621G13443化G2530G17241高G17907离G5527，G11784G3363纳米粒释放其G1025G2265G16077的药物）及G1314G9213高G17907离G5527G8873等。因为乙肝疫苗是G980G12193G15519G11345G12879药物，容易G2476性及吸附，G1000G2010子G18339相对G1122化学药物较大，所以选择G1314G9213高G17907离G5527G8873G8991定纳米粒的G2265G4565G10587及G17745药G18339。G1314G9213高G17907离G5527G8873是G980G12193G8616较G9213和及非G2476性的G7053G8873，G17902G17819较高转G17907的离G5527G17819G12255，使纳米粒G994G7422G2265G16077的药物G6116G2163G2010离，而G2499以设定的G1314G9213环境G1457证G1114乙肝疫苗G3324离G5527G17819G12255G1025不G1262因高G9213而G2476性，离G5527G2530收集纳米粒及上G9177G9094，用HPLCG8991定上G9177G9094G1025乙肝疫苗的G8999G5242，从而计算G2265G4565G10587和G17745药G18339。乙肝疫苗纳米粒的G2265G4565G10587和G17745药G18339见G159321。TABLE1THEENTRAPMENTEFFICIENCYANDLOADINGCAPACITYOFCHITOSANNANOPARTICLESN3HBSAGADDEDGENCAPSULATIONEFFICIENCYHBSAGLOADEDG/MG259231316890124093712317802550927111356046当乙肝疫苗的G8999G5242增G2164G7114，纳米粒G2265G4565G10587并无G7138G7186G2476化，都G332490G705以上（G159321）。12推G7041其原因G2499能是由G1122壳聚糖是G980G12193荷正G11017G3822糖，G4439G3324G994TPP交联G2530G2010子G15932G19766G1185G7099G2109G1325大G18339游离氨基，所以纳米粒G1185然荷正G11017，而乙肝疫苗是G980G12193G17139G11017性G4579G2010子G15519G11345，乙肝疫苗G994壳聚糖首先由G1122静G11017G5353G2159而G13051密结合，G2164入TPPG2530，壳聚糖G2265G16077乙肝疫苗聚集G6116球，两者结合G2159G8616较大，G2265G4565G10587也较高。乙肝疫苗G8999G5242增G2164G7114，相对G1122G2010子G18339较大的壳聚糖来说，G1185然G2499以吸G17745疫苗进G2447，所以G2265G4565G10587G2476化并不G7138G7186。就本G16850G20076来说，并不G16213G8726乙肝疫苗的G8999G5242非常高，而G5062考G