本文選題：雙級靶向 + 阿爾茨海默病�。� 參考：《復旦大學》2014年碩士論文

【摘要】：阿爾茨海默病(Alzheimer's disease, AD)是一種常見的中樞神經系統(tǒng)退行性疾病,其發(fā)病機制復雜,治療難度大,目前臨床上缺乏有效的對因治療藥物。生物技術藥物如神經生長因子、p-片層阻斷肽等,能針對神經細胞的病變原因發(fā)揮作用,在治療AD方面已顯示出良好的應用前景,但要真正發(fā)揮其治療優(yōu)勢還存在兩大難點：首先,藥物難以透過血腦屏障(blood brain barrier, BBB)入腦；其次,藥物入腦后呈全腦分布,對病變部位缺乏選擇性,藥物在發(fā)揮療效的同時,也給正常腦組織帶來了重大安全隱患。因此,如何將治療藥物準確遞送至AD的病變部位,從而增強治療效果,降低中樞副作用,成為AD治療關注的一大重點。為此,本課題采用“雙級靶向”的策略,即在載藥的遞釋系統(tǒng)表面同時修飾雙重靶向功能基,其中第一級靶向功能基能夠選擇性親和BBB,促進遞釋系統(tǒng)跨BBB轉運；入腦后第二級靶向功能基能夠親和病變組織,賦予該系統(tǒng)對病灶組織繼續(xù)尋靶的能力,使更多藥物進一步濃集于腦病變部位,提高治療效果,降低毒副作用。神經細胞外以p-淀粉樣蛋白(β-amyloid, Ap)沉積為核心形成的老年斑是AD的主要病理特征之一,已被廣泛認為是AD最恰當?shù)姆肿由飿擞?是AD診斷和治療的理想靶點。老年斑的主要成分為Aβ42(約占96%),因此,本課題針對BBB和Aβ42沉積形成的老年斑,選擇對其有高親和力的兩個靶向功能基：TGN肽(氨基酸序列為：TGNYKALHPHNG)和QSH肽(氨基酸序列為：QSHYRHISPAQV),將其修飾到聚乙二醇-聚乳酸(PEG-PLA)納米粒表面,構建靶向腦部Aβ42的雙級靶向納米遞釋系統(tǒng),并通過體內外試驗對其雙級靶向性進行確證。進一步包載p-片層阻斷肽H102,考察雙級靶向遞釋系統(tǒng)是否有較單級靶向遞釋系統(tǒng)更優(yōu)的治療AD的效果。本課題研究分為兩部分。第一部分首先構建TGN、QSH修飾的PEG-PLA納米粒(TQNP),并對該納米粒表面TGN和QSH的修飾密度進行優(yōu)化,進一步考察優(yōu)化所得的TQNP的雙級靶向性和細胞攝取機理,并對其細胞毒性進行評價。第一部分第一章采用乳化／溶媒蒸發(fā)法制備PEG-PLA納米粒(NP),將具有腦靶向性的TGN肽和與Aβ42具有高親和性的QSH肽共價連接到納米粒表面,制得雙級靶向納米粒(TQNP)。表面元素分析結果證實,TGN和QSH均被成功修飾到納米粒表面。利用bEnd.3細胞攝取實驗和小鼠活體成像實驗對納米粒表面TGN肽的修飾密度進行優(yōu)化；利用硫磺素-T實驗和表面等離子共振技術(SPR)對納米粒表面QSH肽的修飾密度進行優(yōu)化。結果當納米粒表面馬來酰亞胺基與TGN或QSH的反應摩爾比均為3：1時,修飾密度最佳(T3Q3NP)。第一部分第二章首先對優(yōu)化所得的T3Q3NP的雙級靶向性進行確證。將納米粒與Aβ42以及AD模型小鼠腦切片共孵育,透射電鏡和熒光顯微鏡觀察顯示：T3Q3NP能顯著抑制Aβ42纖維的形成,且與AD模型小鼠海馬淀粉樣斑塊有較強的共定位,表明QSH修飾的納米粒與Aβ42具有較高的親和性。AD模型小鼠尾靜脈給予納米粒后,離體成像定性觀察以及腦分布定量測定均顯示T3Q3NP在Aβ42聚集的海馬區(qū)的濃度顯著高于單功能基修飾和未修飾納米粒,其AUC分別是T3NP和NP的1.61和3.43倍,證實了T3Q3NP具有很好的靶向腦內Aβ42的能力。細胞攝取機制的初步研究表明,雙級靶向納米粒在bEnd.3細胞中的攝取是能量依賴的,且溶酶體和穴樣凹陷均在細胞攝取過程中起重要作用。MTT實驗結果顯示,納米粒在考察濃度范圍(0.1～10 mg/mL)內無明顯細胞毒性,具有較高的安全性。本課題第二部分以p片層阻斷肽H102為模型藥物,制備了載H102的雙級靶向納米粒(T3Q3NP/H102),并對其腦內分布、藥效學以及短期毒性進行了考察。第二部分第一章采用復乳／溶媒蒸發(fā)法制備載H102的納米粒(NP/H102),在納米粒表面共價連接TGN、QSH肽,分別制備得到TNP/H102, QNP/H102和TQNP/H102。制得的四種納米粒粒徑約120 nm, Zeta電位約-28 mV。納米粒的包封率約60%,載藥量約0.55%。納米粒的包載可顯著提高H102在血和腦勻漿中的穩(wěn)定性,載藥納米粒在血漿和PBS中24 h累積釋放百分率分別為65%和39%,顯示出一定的緩釋作用。第二部分第二章對載H102的納米粒的腦內遞藥特性進行考察。建立了H102體內分析的LC-MS方法,經方法學驗證符合要求。AD模型小鼠尾靜脈注射載H102的納米粒(NP/H102、TNP/H102和TQNP/H102),結果顯示,三種納米粒在血中的藥動學基本一致,TNP/H102和TQNP/H102的入腦量均顯著高于NP/H102,且TQNP/H102的海馬靶向指數(shù)DTI為3.67,顯著高于TNP(DTI=1.94),說明TQNP能準確遞送H102至Ap斑塊沉積的海馬區(qū)域。第二部分第三章對H102各制劑的藥效學進行評價。采用小鼠雙側海馬定位注射聚集態(tài)Aβ42構建AD小鼠模型,通過Morris水迷宮實驗評價H102各制劑對AD模型小鼠學習能力及空間記憶障礙的改善作用。結果顯示,TNP/H102和TQNP/H102的改善作用較為明顯,尤其是TQNP/H102高劑量組,小鼠的學習和記憶能力得到明顯提高。生化指標測定以及免疫組化觀察結果可知,TGN修飾納米粒(TNP/H102和TQNP/H102)能明顯抑制Aβ斑塊的沉積,對海馬區(qū)神經細胞具有保護作用,且呈劑量依賴性。其中TQNP/H102中、高劑量能明顯逆轉Aβ42對海馬神經元的損傷。并且,TQNP/H102中劑量組的治療效果與TNP/H102高劑量組相似,表明在相同劑量下,雙級靶向納米粒較單功能基修飾納米粒治療AD作用更優(yōu)。第二部分第四章對TQNP/H102進行了3周的短期毒性考察,結果顯示小鼠的血液學指標正常,各主要臟器(心、肝、脾、肺、腎)均未發(fā)生明顯的組織病理學改變,表明雙級靶向納米粒并未產生體內毒副作用,是一種安全可靠的遞藥系統(tǒng)。
[Abstract]:Alzheimer's disease (Alzheimer's disease AD) is a common neurodegenerative disease of the central nervous system, its pathogenesis is complex, difficult to cure, the lack of an effective clinical treatment for drugs. Biotech drugs such as nerve growth factor, p- layer blocking peptide, can cause the lesions of nerve cells play a role in the treatment of AD, have shown a good prospect, but to really play its advantages there are two major difficulties: first, the drug is difficult to penetrate the blood-brain barrier (blood brain, barrier, BBB) into the brain; secondly, the whole brain was behind the drug distribution, lack of selectivity to the lesion site in the therapeutic effect of drugs at the same time, but also to normal brain tissue brought great security risks. Therefore, how to treat the lesion accurately drug delivery to AD, so as to enhance the therapeutic effect, reduce the central side effects, become AD for attention A major focus. Therefore, this subject adopts "double targeting strategy", namely in the delivery system of drug loading and surface modification of dual targeting functional group, wherein the first level targeting functional group can promote the selective affinity of BBB delivery systems across the BBB transport into the second stage; behind targeting functional group can the affinity of lesions, enabling the system continues to focus on targeting the organization, make further more of the drug concentration in the brain lesions, improve therapeutic effect and reduce side effects. Nerve cells to p- amyloid deposition (beta -amyloid, Ap) to form the core of senile plaque is the main pathological features of AD AD, has been widely considered to be the most appropriate molecular markers, is the ideal target for the diagnosis and treatment of AD. The main component of senile plaques is A beta 42 (approximately 96%), therefore, the topic for the BBB and A beta 42 plaques, choose to have high affinity Two target and force to functional groups: TGN peptide (amino acid sequence: TGNYKALHPHNG) and QSH peptide (amino acid sequence: QSHYRHISPAQV), the modified polyethylene glycol polylactic acid (PEG-PLA) nanoparticles, which target the brain A beta 42 double targeted nano delivery system, and through the body outside test of the two-stage targeting was confirmed. Further p- loaded layer blocking peptide H102 on two level targeting delivery systems if there is a single stage targeting delivery system to better the effect of treatment of AD. This study is divided into two parts. The first part firstly constructed TGN, QSH modified PEG-PLA nanoparticles (TQNP), and on the surface of the nanoparticles modified density of TGN and QSH were optimized to further investigate the optimized TQNP double targeting and cellular uptake mechanism, and to evaluate its cytotoxicity. The first part of the first chapter by emulsification / solvent evaporation method to prepare PEG -PLA nanoparticles (NP), will have the brain targeting peptide TGN and A beta 42 has high affinity for the QSH peptide covalently attached to the surface of the nanoparticles, prepared double targeting nanoparticles (TQNP). The surface elemental analysis results confirmed that TGN and QSH were successfully modified onto the surface of nanoparticles by bEnd.3 cells. Uptake in mice and in vivo imaging experimental density on the surface of TGN nanoparticles modified peptides were optimized; using Thioflavin -T assay and surface plasmon resonance (SPR) density on the surface of QSH nanoparticles modified peptides were optimized. The reaction when the surface of the nanoparticles Moore maleimide groups with TGN or QSH ratio was 3:1, modified the optimum density (T3Q3NP). The first part of the second chapter on the T3Q3NP of the two-stage targeting was confirmed. The nanoparticles with A beta 42 and AD mouse brain slices were incubated, transmission electron microscope and fluorescence microscope observation showed that: The formation of T3Q3NP can significantly inhibit A beta 42 fiber, and AD model of mouse hippocampal amyloid plaques co localization of strong, showed that the nanoparticles modified by QSH and A beta 42 has a higher affinity for.AD mice after intravenous injection of nanoparticles in vitro imaging, qualitative observation and brain distribution determination showed that the concentration of T3Q3NP in the A beta 42 aggregation of the hippocampus was significantly higher than that of single functional groups of modified and unmodified AUC nanoparticles, which are T3NP and NP 1.61 and 3.43 times, confirmed that T3Q3NP has good targeting ability in the brain of A beta 42. Preliminary study on the uptake mechanism showed that double targeting nanoparticles in bEnd.3 cell uptake is energy dependent, and lysosomes and cave in depression play an important role in the cellular uptake of.MTT nanoparticles in the experimental results show that the effects of concentration range (0.1 ~ 10 mg/mL) without obvious cytotoxicity, with high safety All of this topic. The second part of the p layer H102 blocking peptide as a model drug, preparation of H102 loaded double targeting nanoparticles (T3Q3NP/H102), and distribution on the brain, pharmacodynamics and short-term toxicity were investigated. The second part of the first chapter by double emulsion / solvent evaporation nanoparticles prepared by H102 the (NP/H102), in the surface of the nanoparticles covalently connected to TGN, QSH peptides, which were prepared by TNP/H102, QNP/H102 and TQNP/H102. four nanoparticles prepared by about 120 nm in diameter, encapsulation efficiency of the Zeta potential is about -28 mV. nanoparticles of about 60%, the drug loading of about 0.55%. loaded nanoparticles can significantly improve the stability of H102 in blood and brain homogenate, drug loaded nanoparticles in plasma and PBS in the 24 h accumulative release percentage were 65% and 39%, showing a certain sustained-release effect. The second part makes research on the second chapter of the H102 loaded in the brain delivery property. A H102 body LC-MS analysis method, the method validation conforms to the requirements of nanoparticles injected into the tail vein of.AD mice carrying H102 (NP/H102, TNP/H102 and TQNP/H102). The results show that dynamic consistent three kinds of nanoparticles in the blood of the drug, TNP/H102 and TQNP/H102 in the brain were significantly higher than that of NP/ H102, and TQNP/H102 to the hippocampus target index DTI as of 3.67, significantly higher than that of TNP (DTI=1.94), that the hippocampus TQNP can deliver H102 to Ap plaque deposition. Effects of second parts and third chapters on the preparation of H102 learning evaluation. Using the mouse hippocampus injection of aggregation of A beta 42 build AD mouse model, through the evaluation of the Morris water maze effect H102 preparation of AD model mice learning and spatial memory deficits. The results showed that the effect of TNP/H102 and TQNP/H102 is obvious, especially the TQNP/H102 high dose group, the ability of learning and memory in mice To improve observation results and determination. Immunohistochemical and biochemical indexes, TGN modified nanoparticles (TNP/H102 and TQNP/H102) can inhibit the deposition of A beta plaques, has a protective effect on neuronal cells, in a dose-dependent manner. The TQNP/H102 in high doses can significantly reverse A beta 42 on hippocampal neurons injury and the therapeutic effect of TNP/H102 dose group and TQNP/H102 high dose group is similar to that of the same dose, double targeting nanoparticles with single functional group modified nanoparticles on AD effect better. The second part of the fourth chapter investigated the short-term toxicity of 3 weeks of TQNP/H102, showed that mice blood indexes of normal. The main organs (heart, liver, spleen, lung, kidney) showed no apparent histopathologic changes, showed that double targeting nanoparticles did not produce toxic side effects in vivo, is a safe and reliable delivery system.

【學位授予單位】：復旦大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R943;R96

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