【作者】 陈洁；

【导师】 蒋新国； 张奇志；

【作者基本信息】 复旦大学 ， 药剂学， 2011， 博士

【摘要】 血脑屏障的存在限制了大多数具有治疗活性的药物(尤其是多肽蛋白类药物)进入中枢神经系统发挥疗效。鼻脑通路的存在为多肽蛋白类药物避开血脑屏障、直接转运入脑提供了可能。但多肽蛋白药物的透黏膜吸收能力差,易被鼻腔中的酶降解,加上鼻纤毛的清除作用,使得经鼻转运入脑的药量仍然很低,无法达到满意的治疗效果。纳米递药系统能够有效减少多肽蛋白药物的降解,而且有望通过载体的跨膜转运增加药物的吸收。但普通纳米粒仍然存在透鼻黏膜吸收能力有限、鼻腔滞留时间短等局限。凝集素是一类具有高度特异性糖基结合活性的蛋白质或糖蛋白,含有一个或多个可与单糖或寡糖特异且可逆结合的非催化结构域,利用其生物粘附特性可促进黏膜对药物的吸收。本课题选择马铃薯凝集素(Solanum tuberosum lectin, STL)作为靶向功能分子,将其修饰到聚乙二醇-聚(乳酸-羟基乙酸)共聚物(PEG-PLGA)纳米粒的表面,构建一种新型的递药系统(STL-NP)。STL能与鼻腔嗅黏膜上选择性高表达的糖基受体(N-乙酰氨基葡萄糖)特异结合,延长递药系统在鼻黏膜,尤其是嗅黏膜上的滞留时间,诱导黏膜的胞吞转运,增加包载药物经鼻入脑的转运量,以期提高对中枢神经系统疾病的治疗效果,并降低潜在的外周副作用。本课题研究分为两个部分。第一部分首先构建了STL修饰的PEG-PLGA纳米粒(STL-NP),并对该纳米递药系统进行表征。采用Calu-3细胞模型对其体外细胞摄取以及细胞毒性进行评价。通过测定载香豆素-6的NP和STL-NP鼻腔给药后在血和脑组织中的浓度,结合离体成像观察纳米粒鼻腔给药后的脑分布情况,评价该递药系统的脑靶向性,并探索其经鼻入脑的转运通路。第一部分第一章采用复乳/溶媒蒸发法制备了PEG-PLGA纳米粒,利用纳米粒表面的马来酰亚胺基团与巯基化凝集素共价连接,得到马铃薯凝集素修饰的PLGA纳米粒(STL-NP).该纳米粒粒径约110nm,Zeta电位约-30mV。X—射线光电子能谱(XPS)分析结果显示,STL-NP纳米粒表面的主要元素含量发生变化,表明STL连接至纳米粒的表面。红细胞凝集实验证实纳米粒表面连接的凝集素仍保持其凝集红细胞的生物活性,并且该凝集作用可被STL特异性糖基(N-乙酰氨基葡萄糖)抑制。第一部分第二章采用Calu-3细胞模型考察NP和STL-NP的细胞摄取及细胞毒性。结果显示,Calu-3细胞对STL-NP和NP的摄取均呈时间、浓度、温度依赖性。Calu-3细胞对STL-NP有更快的胞吞速度和更强的胞吞能力。特异糖基抑制试验结果显示,STL介导纳米粒与细胞表面糖蛋白的特异性结合是促进细胞摄取的重要条件。胞吞抑制剂的摄取抑制试验结果显示：STL-NP首先通过STL与细胞表面的N-乙酰氨基葡萄糖残基结合促使其吸附在细胞表面；随即细胞通过包被凹陷、穴样凹陷以及巨胞饮途径将纳米粒内吞：入胞后,通过各期内体,部分纳米粒进入溶酶体逐渐降解,而部分纳米粒逃脱溶酶体继续转运或在高尔基体的参与下被胞吐排出细胞;内吞和转运过程是能量消耗的过程。MTT实验结果显示,STL-NP在浓度小于25mg/mL时无细胞毒性,仅在浓度达50mg/mL时显示轻微细胞毒性。蟾蜍上腭离体模型的结果显示,NP和STL-NP对蟾蜍上腭黏膜纤毛无明显毒性。以上结果说明,STL-NP是一种有效且安全的鼻用递药系统。第一部分第三章以香豆素-6为荧光探针定量测定NP和STL-NP鼻腔给药后在血和脑组织中的浓度,结果显示STL修饰后纳米粒的鼻腔吸收和脑转运速度增快,入脑量提高(p<0.01),而吸收入血量降低(p<0.05),对嗅球、大脑和小脑的脑靶向效率分别为NP的1.92、2.45和1.89倍(p<0.01)。以近红外染料DiR为荧光探针进行离体组织脏器的成像观察。结果显示,STL-NP组在0.25～8h时间范围内脑部探针信号均强于NP。STL-NP和NP鼻腔给药后主要分布于肾、肝、脾、肺等脏器。STL-NP鼻腔给药后15min,探针信号即在嗅脑、大脑、脑干等部位广泛分布。嗅球、前嗅核部位的探针信号在2h后逐渐减弱,而脑干、大脑、小脑部位的信号可持续8h以上。提示STL的修饰促进了鼻黏膜对纳米粒的吸收,STL-NP主要沿嗅通路和三叉神经通路转运入脑,其中三叉神经通路的转运更为持久。本课题第二部分以碱性成纤维细胞生长因子(basic fibroblast growth factor, bFGF)为模型药物,制备了载bFGF的STL修饰PLGA纳米粒(STL-bFGF-NP)并进行表征,采用同位素标记法对纳米粒鼻腔给药后脑内递药特性进行考察。采用Aβ25-35和IBO海马区共同注射建立AD大鼠模型,通过Morris水迷宫实验评价bFGF各制剂组对AD模型大鼠空间学习记忆障碍的改善作用,结合生化指标测定和免疫组化研究观察海马区的病理变化。然后对STL-bFGF-NP鼻腔连续给药3周后的短期毒性进行考察,观察鼻黏膜的细胞分化和形态的变化情况,以及对主要脏器(心、肝、脾、肺、肾)的毒性情况。第二部分第一章采用复乳/溶媒蒸发法制备包载bFGF的PLGA纳米粒(bFGF-NP),该纳米粒与巯基化STL共价连接制得STL-bFGF-NP. bFGF-NP和STL-bFGF-NP粒径均为110nm左右,Zeta电位为-30mV左右。STL-bFGF-NP的包封率和载药量较bFGF-NP会有所降低。bFGF-NP在血浆和鼻黏膜匀浆液中的稳定性较bFGF明显提高。bFGF-NP在血浆和鼻洗液中虽有一定的突释(0.5h的释放量分别为11.40%和6.32%),但24h累积释放量分别为73.09%和41.91%,显示出一定的缓释作用。第二部分第二章采用碘标记法对STL-bFGF-NP鼻腔给药后的脑内递药特性及体内分布进行考察。结果显示,bFGF溶液、bFGF-NP和STL-bFGF-NP鼻腔给药后入脑量均较bFGF溶液静脉给药提高,其中STL-bFGF-NP在嗅球、大脑、小脑中的AUC0-24h分别为bFGF溶液静脉给药组、bFGF溶液鼻腔给药组、bFGF-NP组的1.79～5.17倍(p<0.05)、1.61～3.21倍(p<0.05)和1.19～2.06倍(p<0.05),脑靶向效率也分别提高3.61～10.5倍(p<0.01)、1.33～2.69倍(p<0.05)和1.18～2.06倍(p<0.01)。STL-bFGF-NP鼻腔给药后转运到三个脑组织的直接转运百分率均大于70%,尤其对嗅球的直接转运百分率高达90%,说明STL修饰后促进了药物经嗅通路转运入脑。体内分布结果显示,bFGF各制剂组鼻腔给药后在各脏器的分布均显著低于静脉给药组,尤其在肾、肝、脾中分布的减少有利于降低bFGF的外周副作用,提高其临床应用性。bFGF-NP和STL-bFGF-NP组在各脏器中的分布较bFGF溶液鼻腔给药组无显著性差异。第二部分第三章药效学研究结果显示,bFGF各制剂组对AD模型大鼠的空间记忆障碍有不同程度的改善作用,其中STL-bFGF-NP组(20μg/kg/d)显示出更快的学习速度。bFGF-NP高剂量组(40μg/kg/d)和STL-bFGF-NP组在长期和短期记忆考察中均显示出更好的记忆效果。STL-bFGF-NP治疗可降低AD大鼠海马AChE活性,提高ChAT水平至AD模型组的2.04倍,对胆碱能神经纤维具有维持存活和营养保护的作用；且能够减轻Aβ斑块在海马区的沉积减少海马区神经细胞的凋亡,从而对AD模型大鼠海马区神经细胞发挥保护作用。第二部分第四章对STL-bFGF-PLGA纳米粒进行了为期3周的短期毒性考察,结果显示鼻黏膜及心、肝、脾、肺等脏器均未发生明显的组织病理学改变。由于肾脏是bFGF的主要毒性靶器官,鼻腔给药组的变性程度较静脉给药组明显减轻,提示鼻腔给药可减轻药物的肾毒性。更多还原

【Abstract】 While enormous progress has been made regarding our understanding of the pathogenic mechanisms of central nervous system (CNS) disorders, there are few effective drugs for treating these diseases. A key obstacle for developing drugs for treating CNS diseases is the blockage of exogenous substances entrance into the brain by the blood-brain barrier (BBB). Many studies have indicated that intranasal administration could offer noninvasive delivery of large molecules to directly enter into the CNS through olfactory pathways or the trigeminal nerve pathway, effectively bypassing the BBB. However, after administrated nasally, the total amount of these macromolecules accessing the brain was reported to be very low, because of the poor capacity of penetration through nasal mucosa, the enzymatic degradation, limited absorption area and the rapid mucociliary clearance. The encapsulation of peptides and proteins into nanometer-sized particles has been shown to protect them from degradation and facilitate their transport across the mucosal barrier. In spite of various advantages, there remains the following "nose-brain barrier" for nanoparticles:first of all, the penetration of nanoparticles through the tight junctions between the nasal epithelium cells is negligible and the endocytosis of the nanoparticles is limited; secondly, the residence time of nanoparticles in nasal cavity is short (within15to20min) because of the mucociliary clearance, which seemed to be not long enough for complete absorption of the formulations.Lectins are proteins or glycoproteins possessing at least one noncatalytic domain which binds reversibly to specific mono-or oligosaccharides. They specifically recognize and bind with carbohydrate residues on cell surface to initiate vesicular transport processes in cells. Thus, a novel Solanum tuberosum L. modified PLGA nanoparticles (STL-NP) were constructed to enhance the nose-to-brain delivery and decrease the potential peripheral toxicity,.In the first part, STL-conjugated PLGA nanoparticles (STL-NPs) were prepared and characterized. Cellular uptake of STL-NP was quantitatively and qualitatively evaluated using Calu-3cell line as an in vitro nasal epithelial model. The ability of STL-NP to mediate drug delivery to the brain after intranasal administration was subsequently evaluated by near infrared fluorescence probe based ex vivo image as well as quantitative determination of the blood and brain tissue concentrations of coumarin-6, a lipophilic fluorescent probe, associated to STL-NP and NP. At last, the safety of STL-NP was investigated by in vitro cytotoxicity and in situ ciliotoxicity experiments.To achieve this goal, maleimide-PEG-PLGA was blended with MePEG-PLGA to prepare nanoparticles by emulsion/solvent evaporation method. The resulting nanoparticles were surface-modified with STL through covalent coupling reaction of maleimide group with thiol group from thiolated STL. The nanoparticles prepared had an average volume weighted diameter of about110nm, and the Zeta potential were about-30mV. The results of X-ray photoelectron spectroscopy (XPS) analysis showed that the content of main elements on the surface of nanoparticles changed, which indicated that STL had been conjugated to the surface of NPs. Haemagglutination test showed that STL, treated through the covalent coupling procedure, still remain their carbohydrates binding bioactivity on the surface of nanoparticles, which could be specifically inhibited by chitin hydrolysates.Uptake of STL-NP by the Calu-3cells was time-, concentration-and concentration-dependent. STL induced strong mucoadhesion on the surface of Calu-3cells, and then triggered or facilitated the internalization of STL-NP. The preincubation with excess chitin hydrolysates heavily suppressed the uptake of STL-NP at37℃, but have little influence on NP uptake, implying that STL-mediated specific adhesion of STL-NP to cell surface glycoproteins was the requisite first step in the cellular uptake. The results of endocytosis inhibition experiment demonstrated that STL-NPs were endocytosed via an energy-dependent process, involving clathrin, caveolae, macropinocytosis, lysosome and golgi apparatus. NP and STL-NP presented no toxicity on Calu-3cells at the concentration below25mg/mL, while showed mild cytotoxicity after24-hour incubation when the concentration increased to50mg/mL. The STL-NP safety experiments showed mild cytotoxicity and negligible cilia irritation. These intriguing in vitro and in vivo results suggest that STL-NP might serve as a promising and safe drug delivery system for intranasal administration.Following intranasal administration, coumarin-6carried by STL-NP was rapidly absorbed into blood and brain. The AUC(0→12h) of coumarin-6in blood, olfactory bulb, cerebrum and cerebellum were about0.77-,1.48-,1.89-and1.45-fold of those of NP, respectively (p<0.05). STL-NP demonstrated1.89-2.45times (p<0.01) higher brain targeting efficiency in different brain tissues than unmodified NP. Enhanced accumulation of STL-NP in the brain was also observed by near infrared fluorescence probe image following intranasal administration. The kidney, liver, spleen and lung were the main distribution organs of STL-NP after intranasal administration. The fluorescence signal of STL-NP appeared in olfactory bulb, cerebrum and brainstem early at0.25h. The signal in olfactory bulb decreased gradually after2h, while the obvious signal in brainstem, cerebrum and cerebellum lasted for more than8h, which indicated that the trigeminal nervous transport was more persistent.In the second part, the basic fibroblast growth factor (bFGF) was selected as a model drug and incorporated into the STL-NPs. The in vivo distribution and brain delivery characteristics of bFGF were investigated by isotopic labeling method. The AD rat model was established by bilateral injection of Aβ25-35and Ibotenic acid (IBO) into rat hippocampus. The protective effects of STL-bFGF-NP treatment for spatial memory deficits in AD rats was evaluated by Morris water maze experiment. After a3-week continuously intranasal administration of STL-bFGF-NPs, the influence on the morphology and cells differentiation of the nasal mucosa, as well as main organs (heart, liver, spleen, lung, kidney) were examined to evaluate the short-term toxicity.The bFGF-NPs were prepared by double emulsion/solvent evaporation method, followed by surface modification with STL. The mean size of the resulting nanoparticles was about110nm and the zeta potential was-30mV. The encapsulation efficiency and drug loading capacity of STL-bFGF-NP slightly decreased compared with bFGF-NP. The stability of bFGF-NP in rat plasma and rabbit nasal mucosa tissue homogenate was increased obviously. A slight burst of bFGF from bFGF-NP was observed in both plasma and nasal washing fluid (11.40%and6.32%, respectively) in vitro release experiment, while a sustained release was followed (73.09%and41.91%, respectively) after incubation in plasma and nasal washing fluid for24h.bFGF was radioiodinated by iodogen method to study the in vivo distribution of the drug after absorption as well as its ability to be delivered to the brain. The results showed the AUC0-24h of125I-bFGF incorporated in bFGF-NP in olfactory bulb, cerebrum and cerebellum was1.20-1.55times of that treated with bFGF solution after intranasal administration, and the AUC0-24h of125I-bFGF carried by STL-bFGF-NP was increased to1.61-3.21times of that from bFGF solution. STL-bFGF-NP demonstrated1.33-2.69times and1.18-2.06times higher brain targeting efficiency in different brain tissue than bFGF solution and bFGF-NP. bFGF was distributed mainly in the kidney, liver and spleen absorbed into the circulatory system following intranasal administration, and radioactive counts in these organs were significantly decreased in comparison with the intravenous group. The distribution of125I-bFGF in both liver and spleen from bFGF-NP and STL-bFGF-NP showed no significant increase when compared with bFGF solution.The results of Morris water maze experiment indicated that STL-bFGF-NPs (20μg/kg/d, in) treatment showed more better spatial learning ability than bFGF solution (40μg/kg/d, in and iv) and bFGF-NPs (20μg/kg/d and40μg/kg/d, in) in AD rats. And the STL-bFGF-NPs (20μg/kg/d, in) and bFGF-NPs (40μg/kg/d, in) treatment showed significantly improved ability in both short-term and long-term memory. The decreased activity of AChE and improved level of ChAT in the hippocampus of AD rats could be observed by treatment with bFGF. The ChAT level in rats treated with STL-bFGF-NP (20μg/kg/d, in) was2.04-fold of that in AD model control rats. In combination with the immunohistochemisty results in hippocampal sections, bFGF treatment demonstrated a protective effect on the hippocampal cells in AD rats by decreasing the apoptosis of the nerve cells and lowering the deposition of Aβ plaque. The trophic action of bFGF on the central cholinergic system, including elevating the ChAT activity and maintaining cholinergic nerve function, could weaken the cholinergic system dysfunction and cognitive function impairment caused by AD.The short-term toxicity of STL-bFGF-NP was investigated following repeated intranasal administration for three weeks. No obvious histopathological changes was found in the nasal mucosa as well as the main organs (heart, liver, spleen, lung). For the kidney is the target organ for in vivo toxicity of bFGF, less vacuolar degeneration was observed in renal tubular epithelial cells, compared to that of the intravenous administraton of bFGF solution group. These results indicated that the renal toxicity could be obviously alleviated by intranasal administration.更多还原