【作者】 杨安树；

【导师】 杨祥良； 刘卫；

【作者基本信息】 华中科技大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 近年来，高分子纳米载药系统作为多肽、蛋白质药物载体展示了良好的临床应用前景，成为药学领域的研究热点。但是，有关高分子纳米载药系统长循环机制的一些基础科学问题尚待深入研究。本文采用开环聚合法合成了不同分子量的单甲氧基聚乙二醇-聚乳酸羟基乙酸(MePEG-PLGA)两嵌段共聚物，通过自乳化溶剂扩散法(SESD)制备了不同表面特性和粒径MePEG-PLGA纳米粒(MePEG-PLGA-NPs)，较系统的研究了血清蛋白吸附、调理作用与MePEG-PLGA-NPs表面MePEG修饰、粒径之间的关系，巨噬细胞吞噬与MePEG-PLGA-NPs表面MePEG修饰、粒径以及血清蛋白吸附、调理作用之间的关系，以阐明高分子纳米载药系统长循环机制。同时，制备了载TNF-α拮抗肽(TNF-BP)的MePEG-PLGA-NPs，并对其载药性能、体外药物释放行为和生物活性等方面进行了研究。主要研究工作如下：(1)采用开环聚合法合成了MePEG-PLGA两嵌段共聚物，利用凝胶渗透色谱(GPC)测定聚合物分子量，利用FT-IR、~1H-NMR表征共聚物的分子结构。结果表明：合成的聚合物分子量为38000Da，图谱证实合成的MePEG-PLGA两嵌段共聚物，纯度较高。以合成的MePEG-PLGA为原料，通过SESD法制备MePEG-PLGA-NPs，以纳米粒粒径和表面Zeta电位为衡量指标，对影响纳米粒特性的处方进行优化，得到制备纳米粒的优化处方：乳化剂为0．2％Tween 80(w／v)，有机相中MePEG-PLGA浓度为8．0 mg／mL，有机相与外水相体积比为1：6。在该处方下，制备的纳米粒粒径为80．2±7．7 nm，表面Zeta电位为-24．55±3．50 mV，透射电镜(TEM)和原子力显微镜(AFM)观察到纳米粒形态规整，分散性能好，具有很好的稳定性。(2)以不同MePEG-PLGA聚合物为材料制备一系列MePEG-PLGA-NPs，利用BCA蛋白试剂盒分析、SDS-PAGE和ELISA等技术系统研究了纳米粒表面MePEG链长、含量及粒径对MePEG-PLGA-NPs吸附血清蛋白和激活补体的影响。研究表明：纳米粒表面MePEG修饰后并随其链长增加，颗粒表面BSA、IgG和血清总蛋白吸附量减少；MePEG含量由2．5％增至10．0％时，IgGn及附量逐渐下降，而BSA和血清总蛋白在MePEG含量为7．5％时，吸附量最低，随MePEG含量进一步增加吸附量略有升高。纳米粒MePEG修饰后，补体消耗减弱，MePEG含量为5．0-7．5％时，补体消耗程度最低。通过检测补体活化片段(iC3b和C4d)，也证实MePEG修饰能降低纳米粒激活补体的程度，并揭示了纳米粒以经典和旁路两种途径激活补体。MePEG-PLGA-NPs的粒径也影响着血清蛋白在其表面的吸附、调理作用，随着粒径由73．6 nm增至405．1nm，血清蛋白吸附和补体激活增强，当纳米粒粒径大于405．1 nm时，蛋白吸附和补体激活不再有明显变化。(3)利用SESD法制备尼罗红(nile red)标记纳米粒，采用荧光分光光度计和荧光显微镜分析巨噬细胞吞噬特性，研究了纳米粒表面MePEG修饰、粒径和血清蛋白吸附、调理对小鼠腹腔巨噬细胞体外吞噬MePEG-PLGA-NPs的影响。研究发现：巨噬细胞吞噬纳米粒包括不依赖血清介导的吞噬和依赖血清介导的吞噬，MePEG修饰对不依赖血清介导吞噬的影响不明显，而对依赖血清介导吞噬的影响是通过调控IgG、补体(经典和旁路途径)参与的调理作用来实现，随着MePEG链长和含量增加，调理作用对巨噬细胞吞噬的影响逐渐减弱，且细胞吞噬率逐渐降低，当MePEG含量超过7．5％时，MePEG链抑制巨噬细胞吞噬纳米粒的作用不再有明显变化。颗粒粒径在纳米和亚微米范围内，随着粒径增大，不依赖血清介导的吞噬逐渐降低，而依赖血清介导的吞噬增强，这两方面作用使得微粒粒径约为400 nm时，最易被巨噬细胞吞噬。(4)以MePEG-PLGA-NPs为载体，通过静电吸附作用负载TNF-BP，探讨了乳化剂种类、乳化剂浓度、聚合物浓度、投药量和载药介质等因素对MePEG-PLGA-NPs载药性能的影响，并对负载TNF-BP纳米粒释药性能和生物活性进行了研究。结果表明：在优化条件下纳米粒的载药量为1．36±0．05％，包封率为54．40±1．56％。MePEG-PLGA-NPs载药量高，药物释放速率快；释放介质的离子强度低，药物释放速率减慢。MTT检测结果证实纳米粒释放的TNF-BP仍具有较高的生物活性。本文研究结果表明，通过SESD可制得具有良好稳定性的MePEG-PLGA-NPs，纳米粒表面MePEG修饰主要通过调控调理素(IgG和补体)和去调理素(BSA)在纳米粒表面的吸附影响依赖血清介导的细胞吞噬特性，因而对巨噬细胞吞噬纳米粒产生影响；粒径对不依赖血清介导和依赖血清介导的巨噬细胞吞噬均有影响。MePEG-PLGA-NPs具有良好的载TNF-BP性能及一定的缓释作用，释放后药物的生物活性较好。本文研究结果一定程度上揭示了纳米载药系统长循环机制，并为制备具有长循环特性的载多肽蛋白药物纳米载药系统提供实验依据。在本文研究基础上，有必要对长循环纳米载药系统在体内的药物动力学与组织分布规律以及相关药效作进一步研究。更多还原

【Abstract】 Recently,nano drug delivery systems （NDDS） that are made up of biodegradablematerials have been the hotspot research in modern pharmacy.However,to date,somemechanisms involved in the long-circulating NDDS have not been fully understood.Inthis study,a series of methoxypolyethyleneglycol modified poly （D,L-lactide-co-glycolide） nanoparticles （MePEG-PLGA-NPs） were prepared by the spontaneousemulsification solvent diffusion （SESD） method,and the correlations among the surfaceproperty and size of nanoparticles,opsonization by serum protein and phagocytic uptakewere studied to illustrate the long-circulating mechanisms for MePEG-PLGA-NPs.Tumornecrosis factor alpha blocking peptide （TNF-BP） was loaded on MePEG-PLGA-NPs byelectrostatic interaction,and TNF-BP loading capacity,bioactivity and the in vitro drugrelease were studied.The research was mainly concerned with the following aspects.（1） MePEG-PLGA copolymer was synthesized by ring-opening polymerization.Themolecular weight （MW） of the MePEG-PLGA copolymer was determined by GPCchromatograph.The structure of copolymer was confirmed by the Fourier transforminfrared spectrum （FT-IR） and ¹H-NMR spectrum.The results showed that the MW of thecopolymer was about 38000 Da.The blank MePEG-PLGA-NPs were prepared by theSESD method,and the NPs were characterized in terms of particle size,zeta potential andmorphology.The main preparation parameters were optimized in order to obtain theMePEG-PLGA-NPs with the desired characteristics.It obtained the optimal conditions forpreparation of nanoparticles that the emulsifier was Tween 80 （0.2%）,the concentration ofcopolymer in the organic phase was 8.0 mg/ml,and the volume ratio of organic phase toaqueous phase was 1:6.Average size and zeta potential of optimized MePEG-PLGA-NPswere 80.2±7.7 nm and -24.55±3.50 mV,respectively.The morphology ofMePEG-PLGA-NPs was examined by transmission electron microscope （TEM） andatomic force microscope （AFM）,and it appeared that the nanoparticles were spherical inshape with smooth surface and without any aggregation or adhesion.（2） A series of MePEG-PLGA-NPs were prepared.The influences of MePEG chain length,MePEG content and particle size on serum protein adsorption and complementactivation were investigated systematically,and their capacity for adsorbing albumin andthe serum total proteins was measured by a bicinchoninic acid （BCA） protein assay.Theadsorption of serum total IgG was investigated by enzyme-linked immunosorbent assay（ELISA）.The results showed that the longer MePEG chains grafted on NPs surfaces,thelower the protein adsorption including albumin,immunoglobulin G （IgG） and the serumtotal protein adsorption.With the contents of MePEG on NPs surfaces increased from2.5% to 10.0%,a reduction of IgG adsorption was observed.BSA and serum total proteinadsorption were reduced with the increase of MePEG content on NPs surfaces up to 7.5%（w/w）,but further increase led to a slight increase of adsorption.Complement activityperformed by hemolytic CH₅₀ test indicated that PLGA-NPs with MePEG content withinthe range of 5.0-7.5 % induces the lowest of complement consumption.Complementfragments （iC3b and C4d） in serum analyzed by ELISA demonstrated that the MePEGcoating could reduce the complement activation via both the classical and the alternativepathways.For the different size of MePEG-PLGA-NPs,protein adsorption andcomplement activation were increased along with the particle size increased from 73.6 to405.1 nm.However,no significant difference could be observed when particle size above405.1 nm.（3） To study the influence of MePEG modification,particle size and opsonization byserum proteins on uptake efficiency of MePEG-PLGA-NPs by phagocytes,nilered-labeledNPs were prepared by SESD method.The uptake efficiency of NPs by murine peritonealmacrophages （MPM） was measured by fluorescence spectrometer and fluorescentmicroscope.Phagocytic uptake of MePEG-PLGA-NPs by MPM involvedserum-independent and serum-dependent phagocytosis.Only serum-dependentphagocytosis was significantly prevented by MePEG modification on PLGA-NP surfaces,and this effect was mainly ascribed to the reduction of IgG adsorption and complementactivation.The surface modification of PLGA-NPs with higher amount and longer chainof MePEG can efficiently inhibit opsonization by serum and subsequently reduce theuptake by phagocytes.However,the inhibition of phagocytic uptake of NPs by MePEGchain was not obviously enhanced when MePEG₅₀₀₀ content above 7.5%.The particle sizeis another critical parameter for the uptake of NPs by phagocytes.Serum-independent phagocytosis decreased and serum-dependent phagocytosis increased with the increase ofparticle size,which contributed to the highest uptake for the MePEG-PLGA-NPs with sizeabout 400 nm.（4） Tumor necrosis factor alpha blocking peptide （TNF-BP） was loaded onMePEG-PLGA-NPs by electrostatic interaction,and the influences of some factors onentrapment efficiency and drug loading such as the type of emulsifiers,the emulsifierconcentration,the polymer concentration,TNF-BP concentration,the pH and ionicstrength of incubation medium were studied.The results showed that the average drugloading and entrapment efficiency for the optimized NPs were 1.36±0.05% and 54.40±1.56 %,respectively.In vitro release studies showed that the more TNF-BP loaded on NPs,the easier the peptides released,and the peptide release became slower for NPs in lowerionic strength medium.It was confirmed that most of TNF-BP remained active afterreleased in phosphate buffer solution by means of MTT.This study demonstrated that MePEG on PLGA-NPs surfaces only significantlyinhibit serum-dependent phagocytosis,which was mainly ascribed to the reduction of IgGadsorption and complement activation.Serum-independent phagocytosis was decreasedand serum-dependent phagocytosis was increased with the increase of particle size.ForTNF-BP,it was shown that MePEG-PLGA-NPs possessed good drug loading,controlledrelease and stability.In the present study,it illustrated the long-circulating mechanisms ofthe NDDS to some extent,and it can also provide new information for the design of moreefficient NDDS for peptides and proteins in medical applications.Based on the presentwork,it is necessary to investigate the pharmacokinetics and tissue distribution oflong-circulating NDDS and in vivo efficacy assessment in the future.更多还原