叶酸修饰的紫杉醇长循环半固体脂质纳米粒的研究

【作者】 吴丽芳；

【导师】 印春华；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2007， 博士

【摘要】 许多肿瘤细胞膜表面的叶酸受体数量和活性显著高于正常细胞,故可利用叶酸为靶标,结合载药给药系统,通过与叶酸受体特异结合而将药物靶向投递到肿瘤组织。本文以紫杉醇为模型药物,制备了叶酸和PEG修饰的紫杉醇半固体脂质纳米粒（Folate-PEG-SSLN）。此Folate-PEG-SSLN具有一定的长循环和肿瘤靶向作用,是较有前景的肿瘤靶向给药系统。1单甲氧基聚乙二醇—磷脂酰乙醇胺酯（MPEG-PE）的合成与表征选用SC-MPEG法以TEA为催化剂活化MPEG,活化率为96.2%。活化PEG与PE进行连接反应,无水乙醚沉淀得粗产物MPEG-PE,硅胶柱层析分离得到MPEG-PE,得率为54.9%。TLC结果显示MPEG-PE的极性介于SC-PEG和PE之间。自制MPEG-PE的熔程为51.7～64.6℃,与标准品MPEG-PE熔程55.8～63.6℃相近。MPEG-PE的SDS-PAGE迁移率较MPEG的小,表明其分子量比MPEG有所增大。红外谱图显示SC-MPEG在1715cm^-1处有二琥珀酰亚胺基碳酸酯的内酰胺五元环特征峰,而MPEG-PE在该处的吸收消失,出现了1738cm^-1处的酯键特征峰,表明PE的氨基取代了SC-MPEG中的琥珀酰亚胺基。¹H-NMR谱图显示δ=5.34ppm处有SC-MPEG与PE连接反应后产生的-OCONH-基团的质子共振峰。综上所述,反应合成了MPEG-PE。2叶酸—聚乙二醇—磷脂酰乙醇胺（Folate-PEG-PE）复合物的合成与表征利用叶酸、PEG-bis-NH₂和PE为原料,两步反应合成Folate-PEG-PE。无水乙醚沉淀得到粗产物,采用两步Sephadex G-25凝胶过滤层析对其进行分离纯化,凝胶谱图表明Folate-PEG-NH₂、Folate-PEG-Folate和Folate-PEG-PE分离良好。TLC结果显示Folate-PEG-PE收集物为单一斑点,其R_f为0.85。Folate-PEG-PE的红外谱图在1720cm^-1处有明显的酰胺酯键峰,而反应物在此处皆无峰。Folate-PEG-PE的¹H-NMR谱图显示δ=4.21ppm是叶酸分子中Glu残基的α-CH₂,δ=2.48ppm是叶酸分子中Glu残基的β,γ-CH₂,δ=3.64ppm是PEG的-OCH₂CH₂。Folate-PEG-PE的得率约为7.5%。3叶酸修饰的紫杉醇长循环半固体脂质纳米粒的制备及性质研究筛选了高温乳化—低温固化法制备PTX-SSLN的处方和工艺。制备了Blank-SSLN、Soan60-SSLN、Myrj59-SSLN、PEG-PE-SSLN和Folate-PEG-SSLN,由透射电镜可见Span60-SSLN为多边形,其余几种SSLN为圆形,分散较好,且在Myrj59-SSLN、PEG-PE-SSLN和Folate-PEG-SSLN表面可见聚乙二醇“外壳”。包封率均在95%以上。Folate-PEG-SSLN和PEG-PE-SSLN的粒径分别为207.2nm和213.3nm,而Span60-SSLN和Myrj59-SSLN的粒径则分别约为275.5nm和236.7nm,在同样配方和制备工艺条件下,Folate-PEG-SSLN和PEG-PE-SSLN具有更小的粒径,可能是由于MPEG-PE具有更好的乳化作用。几种SSLN都带负电荷,Folate-PEG-SSLN和PEG-PE-SSLN的Zeta电位分别为-34.92mV和-37.54mV,而Span60-SSLN和Myrj59-SSLN则分别为-29.21mV和-28.16mV,Folate-PEG-SSLN和PEG-PE-SSLN的Zeta电位低于Span60-SSLN和Myrj59-SSLN,可能具有更好的稳定性。几种纳米粒的粘度都较小。4叶酸修饰的紫杉醇长循环半固体脂质纳米粒的体外释放及细胞摄取试验Span60-SSLN在6d内药物释放完全,在12d内PEG-PE-SSLN和Myrj59-SSLN分别释放85%和78%,而Folate-PEG-SSLN在15d内释放54%。含PEG链的PEG-PE-SSLN、Myrj59-SSLN和Folate-PEG-SSLN较不含PEG链的Span60-SSLN释药缓慢,可能是由于PEG链能够起到有效的空间位阻作用,可阻止纳米粒子之间的“碰撞”,减少纳米粒的聚集,减缓药物从固体脂质的骨架扩散释放。在24h内Span60-SSLN、PEG-PE-SSLN、Myrj59-SSLN和Folate-PEG-SSLN分别释药43%、29.6%、23.5%和10.7%,表现为24h内释放迅速,此后延迟释药。在30min内,Span60-SSLN、PEG-PE-SSLN和Myrj59-SSLN的MPM摄取率分别为2.31%、0.66%和0.07%。加入血浆后Span60-SSLN、PEG-PE-SSLN和Myrj59-SSLN的MPM摄取率分别为3.2%、1.62%和0.14%,这些结果表明血浆蛋白调理后促进巨噬细胞的吞噬,而PEG链能在一定程度上阻止巨噬细胞对纳米粒的摄取。由于PEG长链的保护,Myrj59-SSLN、PEG-PE-SSLN和Folate-PEG-SSLN体外释放较为缓慢,具有较强的抵抗血浆蛋白吸附和躲避巨噬细胞吞噬的能力,预示其可能具有良好的体内长循环特性。5叶酸修饰的紫杉醇长循环半固体脂质纳米粒大鼠体内药代动力学研究建立了以地西泮为内标的RP-HPLC测定大鼠血浆中紫杉醇浓度的方法。大鼠尾静脉注射Folate-PEG-SSLN、PEG-PE-SSLN和Span60-SSLN,给药后24h,Span60-SSLN组大鼠体内药物浓度低于检测限,而PEG-PE-SSLN和Folate-PEG-SSLN组大鼠体内药物浓度分别为375ng·mL^-1和258.7ng·mL^-1。平均血药浓度-时间曲线显示含有PEG链的紫杉醇SSLN血中药物浓度下降趋势比未含PEG链的SSLN有所减慢。Folate-PEG-SSLN和PEG-PE-SSLN的AUC增加,体内滞留时间MRT和t_1/2β延长,表明制备的Folate-PEG-SSLN和PEG-PE-SSLN具有一定长循环特性,有可能更好的靶向肿瘤部位。6叶酸修饰的紫杉醇长循环半固体脂质纳米粒在S-180荷瘤小鼠体内的组织分布及抑瘤率试验在0.25h到12h内,不同时间点Folate-PEG-SSLN和PEG-PE-SSLN组在肿瘤内的药物分布百分率都高于溶液剂组,提示其有一定的肿瘤靶向性。在0～12h内,与溶液剂组相比,Folate-PEG-SSLN组肿瘤AUC_0-12提高了25%。Folate-PEG-SSLN和PEG-PE-SSLN的肺AUC分别减少86.5%和90.9%;在肝脏中的AUC分别减少36.1%和70.6%,在一定程度上减少肝巨噬细胞的吞噬;在心脏中的AUC分别减少94%和78.9%,提示Folate-PEG-SSLN和PEG-PE-SSLN可能具有降低心脏毒性的作用;在血浆中的AUC分别是溶液剂的2.47和2.33倍,表明Folate-PEG-SSLN和PEG-PE-SSLN具有一定的长循环特性,有可能更多的靶向肿瘤部位。PEG-PE-SSLN和Folate-PEG-SSLN组肿瘤的相对摄取率（Re）分别为0.85和1.25,表明Folate-PEG-SSLN对肿瘤有一定靶向性。PEG-PE-SSLN和Folate-PEG-SSLN的靶向效率都小于1,表明其组织选择性差。PEG-PE-SSLN和Folate-PEG-SSLN对紫杉醇溶液剂的肿瘤相对靶向效率分别是5.26和5.77,显示比溶液剂有更好的肿瘤靶向性,且靶向性Folate-PEG-SSLN略好于PEG-PE-SSLN。同剂量的Folate-PEG-SSLN和溶液剂的抑瘤率分别为44.38%和37.32%,Folate-PEG-SSLN组略高于溶液剂组。组织形态学观察显示Folate-PEG-SSLN(6mg·kg^-1)能明显抑制肿瘤细胞的生长。7叶酸修饰的紫杉醇长循环半固体脂质纳米粒冻干粉的制备及稳定性初步研究筛选出以5%海藻糖为冻干保护剂。以5%海藻糖为冻干保护剂时,PEG-PE-SSLN和Folate-PEG-SSLN能保持较好的形态和分散性,粒径增大较少,Zeta电位绝对值都略有降低。DSC结果显示,制备成SSLN后,紫杉醇以无定形态存在于SSLN中;¹H-NMR也表明脂质和药物紫杉醇在SSLN中的结构状态发生了改变,具有新的热力学性质。稳定性初步试验发现,本文制备的SSLN冻干粉宜于在4℃贮存。在4℃条件下贮存3个月后,其外观、粒径和包封率变化都较小,且分散性良好。更多还原

【Abstract】 The number and activity of folate receptors on the membrane surface of tumor cells are significantly higher than that of normal cells.Hence,folate can be used together with drug delivery system to achieve the targeting delivery of drug to the tumor tissues by the specific interaction between folate and receptors.In this study, paclitaxel（PTX） is used as a model drug,and PTX semi-solid lipid nanoparticles （SSLN） modified with folate and PEG are prepared.This kind of SSLN with certain long-circulating and tumor-targeting functions is a promising tumor-targeting drug delivery system.1 Preparation and characterization of monomethoxypolyethylene glycol-phosphatidylethanolamine（MPEG-PE）SC-MPEG with activity degree of 96.2%is prepared using TEA as the catalyst. The activated PEG is allowed to react with PE,followed by addition of anhydrous ethyl ether to precipitate MPEG-PE.The coarse MPEG-PEG is purified using silica gel chromatography with a yield about 54.9%.Result of TLC shows that the polarity of MPEG-PE is between SC-MPEG and PE.The melting range of the self-prepared MPEG-PE is 51.7-64.6℃,which is similar to that of the standard substance of MPEG-PE（55.8-63.6℃）.The electrophoretic mobility ratio of MPEG-PE in SDS-PAGE gel is lower than that of MPEG.The FTIR spectrum of SC-MPEG displays a peak at 1715cm^-1 which is specific for the inner lactam.MPEG-PE has a FTIR peak of ester bond at 1738cm^-1 instead of the peak at 1715cm^-1,which shows that the amino group of PE has displaced the succinimide group in SC-MPEG.The ¹H-NMR spectrum of MPEG-PE exhibits a chemical shift at 5.34ppm that is induced by the hydrogen of amide linkage in MPEG-PE.As stated above,MPEG-PE has been prepared.2 Preparation and characterization of the folate-PEG-PE compositeFolate-PEG-PE is synthesized with PEG-bis-NH₂ and PE through two-stepped reactions.The reaction mixture is dealt with anhydrous ethyl ether to precipitate folate-PEG-PE,and the coarse product is further purified by Sephadex G-25 gel filtration chromatography.It is showed that folate-PEG-NH₂,folate-PEG-folate and folate-PEG-PE are well separated from each other.TLC of the fraction of folate-PEG-PE displays a single spot with R_f of 0.85.There is a strong peak at 1720cm^-1 in the FTIR spectrum of folate-PEG-PE,which is specific for amide linkage, while all the reactants have no such peak.The ¹H-NMR spectrum of folate-PEG-PE exhibits chemical shifts at 4.21ppm,2.48ppm and 3.64ppm that can be ascribed toα-CH₂,β,γ-CH₂ in Glutamate moiety of folate and the repeated units of PEG, respectively.In summary,folate-PEG-PE has been prepared using the above method with a yield of 7.5%.3 Preparation and characterization of PTX loaded SSLNThe formula and technology for preparation of PTX-SSLN by the procedure of "emulsification at a high temperature and solidification at a low temperature"are established in this study.Five kinds of SSLN including blank-SSLN,Span60-SSLN, Myrj59-SSLN,PEG-PE-SSLN and folate-PEG-SSLN are prepared.Span60-SSLN appears to be polygon under TEM,and the other kinds of SSLN are in round shape with good dispersion.Besides,the PEG "shells"can be observed on the surface of Myrj59-SSLN,PEG-PE-SSLN and Folate-PEG-SSLN.The entrapment efficiencies of these SSLNs are all over 95%.The mean particle sizes of folate-PEG-SSLN, PEG-PE-SSLN,Span60-SSLN and Myrj59-SSLN are 207.2nm,213.3nm,275.5nm and 236.7nm,respectively.The particle sizes of folate-PEG-SSLN and PEG-PE-SSLN are smaller than that of Span60-SSLN and Myrj59-SSLN under the same preparing conditions,which can be attributed to the better emulsification effect of MPEG-PE.All the five SSLNs are negatively charged.The Zeta potentials of folate-PEG-SSLN,PEG-PE-SSLN,Span60-SSLN and Myrj59-SSLN are -34.92mV, -37.54mV,-29.21mV and -28.16mV,respectively.The absolute value of Zeta potentials of folate-PEG-SSLN and PEG-PE-SSLN are higher than that of Span60-SSLN and Myrj59-SSLN,which indicate that the former two SSLN may hold more stability.In addition,the viscosities of all the five SSLNs are low.4 In vitro drug releasing and cell phagocytosis experiments of PTX loaded SSLNAlmost all the drug is released from Span60-SSLN in 6d.The drugs released from PEG-PE-SSLN and Myrj59-SSLN in 12d are both over 85%and 78%, respectively,and the drug released from folate-PEG-SSLN in 15d exceeds 54%.The drug released from Span60-SSLN is much faster than that from PEG-PE-SSLN, Myrj59-SSLN and folate-PEG-SSLN,which attached to PEG chains.The stereo hindrance effect of PEG chains prevents collisions between nanoparticles and therefore decreases drug releaseing by diffusing through the solid lipid matrix.The drugs quickly release from Span60-SSLN,PEG-PE-SSLN,Myrj59-SSLN and Folate-PEG-SSLN during 24h,which are 43%,29.6%,23.5%and 10.7%, respectively.After that,the drug release becomes slower.The MPM uptake rates of Span60-SSLN,PEG-PE-SSLN and Myrj59-SSLN in 30min are 2.31%,0.66%and 0.07%,respectively.After adding blood plasma,the intake rates become 3.2%,1.62%and 0.14%,respectively.This result demonstrates that opsonization by plasma proteins can promote the phagocytosis of nanoparticles by macrophage,while PEG can interfere with the uptake process.5 Pharmacokinefics study of PTX loaded folate-PEG-SSLN in ratsThe analytical RP-HPLC method is established by using Diazepam as the internal standard to determine the concentration of PTX in rats’ plasma.The folate-PEG-SSLN,PEG-PE-SSLN and Span60-SSLN are intravenously administered through tail vein of rats.24h after administration,the PTX concentration in plasma is below the limit of determination for the group of Span60-SSLN,while for the groups of PEG-PE-SSLN and folate-PEG-SSLN,the PTX concentration in plasma are 375ng·mL^-1 and 258.7ng·mL^-1,respectively.The mean plasma concentration of PTX-time curves show that the decreasing rate of plasma concentration of PTX is slower in PEG-PE-SSLN and folate-PEG-SSLN groups with PEG than in Span60-SSLN group without PEG.The increase of the AUC of folate-PEG-SSLN and PEG-PE-SSLN and the elongation of MRT and t_1/2β indicate that folate-PEG-SSLN and PEG-PE-SSLN possess long-circulating effect in some degree,which maybe do favor to target tumor sites.6 The tissue distribution and tumor inhibition rate of PTX loaded folate-PEG-SSLN in S-180 bearing miceThe drug distribution rates of the groups of folate-PEG-SSLN and PEG-PE-SSLN in tumor are higher than that of the group of PTX solution at different time intervals from 0.25h to 12h,which suggests that the two SSLN have certain tumor targetability.Comparing with the PTX solution,the tumor AUC_0-12 of folate-PEG-SSLN group increases by 25%.And the lung AUC₉0-12) of folate-PEG-SSLN and PEG-PE-SSLN groups decreases by 86.5%and 90.9%, respectively.The liver AUC_0-12 decreases by 36.1%and 70.6%,respectively,which indicate that the phagocytosis of folate-PEG-SSLN and PEG-PE-SSLN by liver macrophage decreases in some degree.The heart AUC_0-12 decrease by 94%and 78.9%,respectively,which probably indicate that the toxicities of folate-PEG-SSLN and PEG-PE-SSLN to heart are lower.The plasma AUC_0-12 of folate-PEG-SSLN and PEG-PE-SSLN respectively are 2.47 and 2.33 times of that of PTX solution,which demonstrate that they have certain long-circulating characteristics and can be targeted to tumor tissues much more.The relative uptake rates of folate-PEG-SSLN and PEG-PE-SSLN by tumor tissues are 1.25 and 0.85,respectively,which demonstrate that folate-PE-SSLN has certain tumor targetability.The target efficiencies of folate-PEG-SSLN and PEG-PE-SSLN are less than 1,which shows their poor tissue selectivity.The relative tumor target efficiencies of folate-PEG-SSLN as well as PEG-PE-SSLN to PTX solution are 5.77 and 5.26,respectively,which displays that the two SSLN have better tumor targetability than PTX solution,and the tumor targetability of folate-PEG-SSLN is slightly better than that of PEG-PE-SSLN.The tumor inhibition rates of folate-PEG-SSLN and PTX solution in the same dose are 44.38%and 37.38%,respectively.The histomorphological study shows that folate-PEG-SSLN administered in the dose of 6mg·kg^-1 can evidently inhibit the growth of tumor cells.7 Preparation and the preliminary stability study of lyophilized powder of PTX loaded folate-PEG-SSLNThe trehalose is selected as the freeze-drying protectant.The folate-PEG-SSLN and PEG-PE-SSLN can keep in good morphology and dispersion using 5%trehalose solution as protectant.There is slight increase in particle sizes as well as slight decrease in Zeta potentials of the SSLN.Results of DSC show that PTX presents as amorphous state in the SSLN.Results of ¹H-NMR also demonstrate that some structural changes of the lipids and PTX take place in the SSLN,which displays new thermodynamic properties.The preliminary stability study demonstrates that the SSLN should be stored at 4℃.After storing at 4℃for 3 months,the morphology, particle size and encapsulation efficiency of the SSLN change a little,and the dispersion of the SSLN retains good.更多还原