脑靶向树枝状高分子纳米基因递释系统的研究
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摘要
基因治疗是许多脑部疾病极具潜力的治疗手段。由于治疗基因本身、病毒载体或未经修饰的非病毒载体无法自主跨越血脑屏障(BBB)到达脑部,目前脑部基因导入的主要方法是脑实质直接定位注射,该方法伤害大,难以实施多次给药,且基因产物蛋白很难转运到脑的广泛范围。特异性修饰的非病毒载体有可能经血管途径给药,以非侵袭性方式实现外来基因药物在脑部的广泛表达,但其基因转染和表达的效率普遍较低。探索具有脑靶向性且高效表达的非病毒基因载体,实现经血管给药达到脑部外源性基因的广泛表达,成为目前脑部疾病基因治疗领域的重点和难点之一。
本课题通过药剂学、高分子学和生物学手段的交叉应用,对载体高分子、脑靶向头基进行比较和筛选后最终构建了一种新型高效的脑靶向非病毒纳米基因递释系统。该系统以阳离子高分子聚酰胺-胺(PAMAM)为基础载体高分子,通过亲水性高分子聚乙二醇(PEG)连接新型脑靶向头基乳铁蛋白(Lf),与基因复合形成PAMAM-PEG-Lf/DNA纳米粒。该纳米粒具有以下两大特性:(1)利用脑部表达Lf相关受体的特征,采用Lf作为脑靶向头基修饰载体高分子,增加纳米粒的脑靶向性;(2)所采用的基因载体PAMAM是近年来发展起来的新型阳离子高分子材料,易于修饰,载基因能力较高。
本文第一章首先进行高分子材料的筛选,比较了壳聚糖(CH)、聚乙烯亚胺(PEI)和不同代数PAMAM的基因包载效率、不同纳米粒体外转染原代培养脑毛细血管内皮细胞(BCECs)的效率等性质。琼脂糖凝胶电泳和DNA含量测定(PicoGreen分析)结果显示,各载体高分子与DNA的质量比达到一定值时,各载体高分子可完全包封DNA,复合形成的纳米粒均有一定抵抗外界阴离子物质的置换作用,能保护所包载DNA免受DNase的降解并保持DNA的完整性。荧光显微镜和荧光素酶定量分析结果显示,第5代PAMAM(PAMAM G5)/DNA纳米粒的表达效率较高。综合文献报道,PAMAM G5可较好平衡基因转染效率、细胞毒性等各方面因素,本课题最终采用PAMAM G5为高分子载体。
选定了基础载体高分子后,本文第二章先采用经典脑靶向头基转铁蛋白(Tf)通过PEG修饰PAMAM,构建了PAMAM-PEG-Tf。UV-vis、~1H-NMR和SDS-PAGE结果显示载体高分子PAMAM-PEG-Tf合成成功。荧光显微镜和流式细胞仪检测细胞内摄取纳米粒的结果表明,PAMAM-PEG-Tf的细胞摄取效率在较低浓度范围内呈浓度依赖性,在高浓度时有饱和趋势。并且,~(125)I放射性标记PAMAM后各载体高分子的体内分布结果显示,经Tf修饰后的载体高分子在脑组织的分布量约为单纯PAMAM的2.25倍。制备的PAMAM-PEG-Tf/DNA纳米粒,其粒径在200 nm左右,zeta电位在13 mV左右,电镜观察显示纳米粒呈球形,表面圆整光滑,具有良好的稳定性。经Tf修饰后,纳米粒在BCECs和脑内的表达均显著高于未修饰的纳米粒。但是,Tf作为脑靶向头基存在一定局限性,如Tf受体介导的双向跨BBB转运功能可能降低通过Tf受体介导入脑的纳米粒在脑内的浓集量、生理状态下BBB上大部分Tf受体可能被内源性Tf占据而降低纳米粒脑靶向性等,需要寻找新的高效的脑靶向头基用于基因递释系统的构建,提高载基因纳米粒的脑靶向性和脑内基因表达效率。
根据文献报道,Lf具有优于Tf和抗Tf受体单克隆抗体OX26的脑内转运效率,提示Lf有可能成为更好的脑靶向头基。但目前关于脑部Lf受体的表征主要是在体外BBB模型上,尚无关于脑组织Lf受体表征的报道,本文第三章对原代培养BCECs和小鼠脑组织上的Lf受体进行表征。共聚焦显微镜和结合动力学实验结果显示,Lf与其受体的结合具有饱和性和竞争抑制现象。结合饱和实验结果显示,脑部Lf受体存在高低两个结合位点,低浓度时主要和高结合位点结合,高浓度时才进一步与低结合位点结合。Lf与BCECs表面高低结合位点的Kd值分别为6.77 nM和4815 nM;与脑细胞膜上高低结合位点的Kd值分别为10.61 nM和2228 nM。
综合第三章研究结果和相关文献可见,Lf作为脑靶向头基具有多项优势,包括:Lf受体介导的跨BBB单向转运功能更利于Lf修饰的纳米基因递释系统在脑组织浓集、生理状态下血循环中Lf的低浓度使内源性Lf不会大量占据脑细胞膜上Lf相关受体等。本文第四章首次采用Lf为脑靶向头基,考察Lf修饰的载体高分子PAMAM-PEG-Lf及其载基因纳米粒的体内外性质。荧光显微镜和流式细胞仪检测结果显示,PAMAM-PEG-Lf的细胞摄取效率在一定范围内呈浓度依赖性,在高浓度时有饱和趋势。并且,在相同浓度比较时,PAMAM-PEG-Lf的细胞摄取和脑内分布高于PAMAM-PEG-Tf,而其他组织的分布量显著下降。在此基础上制备了PAMAM-PEG-Lf/DNA纳米粒,其粒径在210 nm左右,zeta电位在25 mV左右,电镜观察显示纳米粒呈球形,表面圆整光滑,同时透射电镜下观察到纳米粒表面的胶体金颗粒,证实了纳米粒表面存在数十个Lf分子。与Tf修饰的纳米粒比较,经Lf修饰后的纳米粒在BCECs和小鼠脑内的表达均显著提高。纳米粒体内基因表达分布结果显示,PAMAM-PEG-Lf/DNA纳米粒的脑内基因表达量约为PAMAM-PEG-Tf/DNA纳米粒的2.3倍,而全身其他器官的基因表达降低,显示了Lf作为脑靶向头基的优越性。
由于Lf首次作为脑靶向头基、载体高分子PAMAM-PEG-Lf为本课题首次合成,国内外未见报道,该载体高分子及其载基因纳米粒的入脑机制尚不清楚,本文第五章对此进行了探讨。细胞摄取抑制实验结果表明,PAMAM-PEG-Lf保留了PAMAM的高分子特性和Lf的配体-受体结合性质,可以经多种途径包括网格蛋白有被小窝和细胞膜穴样内陷依赖性内吞途径以及巨胞饮途径被原代培养BCECs摄取;PAMAM-PEG-Lf载体高分子及其载基因纳米粒主要通过受体介导的穿细胞转运过程跨越BBB,但仍保留了吸附介导的转运机制。另外,经叶绿素铜脑内示踪实验证实PAMAM-PEG-Lf/DNA纳米粒可跨越BBB进入脑组织。
最后,因文献报道帕金森病(PD)患者脑部神经元和微血管上Lf受体表达显著增加,本文第六章以PD为疾病模型,评价了PAMAM-PEG-Lf包载编码人源性胶质细胞源性神经营养因子(GDNF)的治疗基因(hGDNF)的纳米粒在急性、慢性PD模型大鼠上的药效,设计了多次给药方案,并比较了单次、多次静脉给药后的治疗效果,未见文献报道。结果表明,单次给药时,PAMAM-PEG-Lf/hGDNF纳米粒的脑部基因表达最高,且随时间会降低;多次给药可以持续增加纳米粒的脑内基因表达,5次给药的表达量显著高于单次给药或3次给药,证实了多次给药的可行性和有效性。同时,在6-OHDA单侧损毁急性模型上,多次给药组能显著改善阿朴吗啡诱导的PD症状,明显增加纹状体和黑质中多巴胺能神经元的数目,同时还提高了损毁侧多巴胺(DA)及其代谢产物的量,接近未损毁侧的水平,显示出其优越性。在鱼藤酮慢性模型上,多次给予PAMAM-PEG-Lf/hGDNF纳米粒同样显示出优于单次给药的疗效,多次给药能显著改善PD大鼠的行动能力,提高酪氨酸羟化酶(TH)免疫反应性,同时提高纹状体中DA及其代谢产物的水平。以上实验证明,PAMAM-PEG-Lf/DNA纳米粒是一种高效的非病毒载基因系统,多次静脉注射是一种有效可行的非侵袭性给药方式,可持续提高非病毒载体的脑内基因表达。
Gene therapy offers a promising cure for various brain disorders.However, therapeutic genes themselves,viral vectors and unmodified non-viral vectors can not reach the brain following an intravenous administration due to the presence of blood-brain barrier(BBB).Most genes or current vectors must be given via parenchymal injections which are considered to be highly invasive and unable to deliver genes products to global areas of the brain.Non-viral vectors modified with specific proteins might enable widespread expression of exogenous genes throughout the brain following a non-invasive transvascular pathway.The main problem is the low transfection and expression efficiency.Therefore,developing a brain-targeting and efficient non-viral gene vector to enable the global gene expression in the brain is currently one of the focuses in the field of brain gene therapy.
In this study,a novel efficient brain-targeting non-viral nano-scaled gene delivery system was finally constructed with means of pharmaceutics, macromolecular chemistry and biology.Polyamidoamine(PAMAM) was applied as the main macromolecular gene vector.And lactoferrin(Lf),for the first time,was investigated as a brain-targeting ligand in the design of PAMAM-based nanoparticles (NP) to the brain,using polyethyleneglycol(PEG) as a spacer.There are two important attributes of Lf-modified PAMAM-based nanoparticles (PAMAM-PEG-Lf/DNA NP).They are(1) modified with Lf to target the corresponding receptors in the brain to enhance the brain-targeting ability,(2) employing novel cationic macromolecular material,PAMAM,as the main gene vector,with high gene encapsulation ability
The first part compared the in vitro characteristics including gene incorporation efficiency and transfection efficiency in brain capillary endothelial cells(BCECs) of several cationic vectors.The results of agarose gel electrophoresis and PicoGreen assay showed that all the prepared nanoparticles could effectively encapsulate DNA and protect themselves from the displacement of anionic substances and the digestion of DNase I.The results of fluorescent microscopy and luciferase assay indicated that PAMAM G5(the 5~(th) generation of PAMAM) had a reasonable gene expression. Given both the literature and our results,PAMAM G5 could balance the gene transfection efficiency and cytotoxicity,and thereafter was chosen as the main macromolecular vector in the following studies.
Next,transferrin(Tf) was selected as a brain-targeting ligand,conjugated to PAMAM via bifunctional PEG,yielding PAMAM-PEG-Tf.The results of UV-vis, ~1H-NMR and SDS-PAGE demonstrated the successful synthesis of PAMAM-PEG-Tf. This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,PAMAM-PEG-Tf showed 2.25-fold brain uptake compared to PAMAM in vivo.The PAMAM-PEG-Tf/DNA NP has a particle size around 200 nm and zeta potential around 13 mV.It is spherical and has regular shape under the examination of transmission electron microscope.The modification of Tf significantly enhanced the gene expression of PAMAM-PEG-Tf/DNA NP compared to unmodified nanoparticles in both BCECs and mouse brains.However,there exist some shortcomings of Tf as a brain-targeting ligand.For example,the Tf receptor-mediated bidirectional transcytosis of Tf through the BBB might decrease the brain accumulation of Tf-modified nanoparticles,and most of Tf receptors might be occupied by endogenous Tf under physiological conditions so to reduce the brain-targeting ability of Tf-modified nanoparticles.Consequently,it is necessary to find a novel efficient brain-targeting ligand for construction of brain gene delivery systems,then to enhance the brain-targeting ability and brain gene expression efficiency.
Several lines of evidence demonstrated that Lf had higher brain transport than Tf and a mouse monoclonal antibody against rat Tf receptor(OX26),which suggested that Lf might be a potential brain-targeting ligand.Up to now,there has been little information about the characteristics of Lf receptors in the brain tissue.The third part was to characterize the Lf receptors in the BBB and brain tissue of mice.The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells.The time course studies demonstrated the saturation phenomenon of binding between Lf and its receptors.Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs(high affinity:dissociation constant(K_d)=6.77 nM; low affinity:K_d=4815 nM) and membrane preparations of mouse brains(high affinity:K_d=10.61 nM;low affinity:K_d=2228 nM).
According to the above-mentioned results and the literature,Lf has several advantages when being a brain-targeting ligand.For example,the Lf receptor-mediated unidirectional transcytosis of Lf through the BBB might increase the brain accumulation of Lf-modified nanoparticles,and most of Lf receptors might not be occupied by endogenous Lf under physiological conditions.Therefore,Lf was,for the first time,exploited as a novel brain-targeting ligand here to synthesize PAMAM-PEG-Lf,and the in vitro and in vivo characteristics of the macromolecular vector and its DNA-loaded nanoparticles were evaluated in the fourth part.This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,at the same concentration, PAMAM-PEG-Lf showed higher cellular uptake,higher brain accumulation and lower distribution in peripheral tissues,compared to PAMAM-PEG-Tf.The PAMAM-PEG-Lf/DNA NP has a particle size around 210 nm and zeta potential around 25 mV.It is spherical and has regular shape under the examination of transmission electron microscope.Moreover,the observation of colloidal gold on the surface of PAMAM-PEG-Lf/DNA NP demonstrated the successful conjugation of several Lf on the nanoparticle.The modification of Lf significantly enhanced the gene expression of PAMAM-PEG-Lf/DNA NP compared to Tf-modified nanoparticles in both BCECs and mouse brains.The results of quantitative examination of luciferase showed that the brain gene expression of PAMAM-PEG-Lf/DNA NP was about 2.3-fold than that of PAMAM-PEG-Tf/DNA NP,when the gene expression in peripheral tissues markedly reduced.All the results provide evidence that Lf is a promising ligand for the design of gene delivery systems targeting to the brain.
Because Lf was first exploited as a brain-targeting ligand and PAMAM-PEG-Lf was first synthesized in this study,the brain entry mechanisms of this vector and its DNA-loaded nanoparticles remain unclear.These researches were carried out in the fifth part.The results of cellular inhibition showed that PAMAM-PEG-Lf maintained the macromolecular features of PAMAM and the ligand-receptor binding attribute of Lf,and could be taken up by BCECs via multiple pathways including clathrin-,caveolae-dependent endocytosis and macropinocytosis.PAMAM-PEG-Lf and its DNA-loaded nanoparticles could cross the BBB mainly through a receptor-mediated manner,and reserved the adsorptive mediated pathway as well. In addition,the observation of copper chlorophyll under analytical transmission electron microscope demonstrated that PAMAM-PEG-Lf/DNA NP could cross the BBB and reach the brain parenchyma successfully.
As known,an overexpression of Lf receptors was observed in both microvessels and neurons of the substantia nigra in patients with Parkinson's disease(PD).The acute and chronic rat PD models were chosen in the sixth part to evaluate the pharmacodynamics of Lf-modified nanoparticles encapsulating human glial cell line-derived neurotrophic factor gene(hGDNF).And a multiple dosing regimen was for the first time designed to compare the therapeutic effects of single and multiple dosing administrations.The results of single injection showed that PAMAM-PEG-Lf/hGDNF NP had higher brain gene expression compared to other types of nanoparticles,and this gene expression decreased with time.Multiple dosing administrations could enhance and maintain the brain gene products in a higher level.The gene expression of five dosing administrations was significantly higher that that of three dosing administrations.These results demonstrated the feasibility and effectiveness of multiple dosing administrations.In the 6-hydroxydopamine(6-OHDA)-lesioned acute PD model,multiple dosing administrations of PAMAM-PEG-Lf/hGDNF NP could markedly reduce the apomorphine-induced rotations,significantly enhance the number of dopaminergic neurons in striatum and substantia nigra,and increase the content of dopamine(DA) and its metabolites in the lesioned side of rats.In the rotenone chronic PD model, multiple dosing administrations also exhibited better therapeutic effects than single injection.Extending the administration period of PAMAM-PEG-Lf/hGDNF NP could significantly improve locomotor activity,reduce dopaminergic neuronal loss and enhance monoamine neurotransmitter levels on PD rats,which indicating more powerful neuroprotective effects of multiple dosing administrations of Lf-modifined nanoparticles.All the results clearly demonstrate that PAMAM-PEG-Lf/DNA NP is an efficient non-viral gene delivery system,and multiple dosing intravenous administrations could be used as a practical non-invasive means to enhance and maintain the higher brain gene expression of non-viral gene vectors.
本课题通过药剂学、高分子学和生物学手段的交叉应用,对载体高分子、脑靶向头基进行比较和筛选后最终构建了一种新型高效的脑靶向非病毒纳米基因递释系统。该系统以阳离子高分子聚酰胺-胺(PAMAM)为基础载体高分子,通过亲水性高分子聚乙二醇(PEG)连接新型脑靶向头基乳铁蛋白(Lf),与基因复合形成PAMAM-PEG-Lf/DNA纳米粒。该纳米粒具有以下两大特性:(1)利用脑部表达Lf相关受体的特征,采用Lf作为脑靶向头基修饰载体高分子,增加纳米粒的脑靶向性;(2)所采用的基因载体PAMAM是近年来发展起来的新型阳离子高分子材料,易于修饰,载基因能力较高。
本文第一章首先进行高分子材料的筛选,比较了壳聚糖(CH)、聚乙烯亚胺(PEI)和不同代数PAMAM的基因包载效率、不同纳米粒体外转染原代培养脑毛细血管内皮细胞(BCECs)的效率等性质。琼脂糖凝胶电泳和DNA含量测定(PicoGreen分析)结果显示,各载体高分子与DNA的质量比达到一定值时,各载体高分子可完全包封DNA,复合形成的纳米粒均有一定抵抗外界阴离子物质的置换作用,能保护所包载DNA免受DNase的降解并保持DNA的完整性。荧光显微镜和荧光素酶定量分析结果显示,第5代PAMAM(PAMAM G5)/DNA纳米粒的表达效率较高。综合文献报道,PAMAM G5可较好平衡基因转染效率、细胞毒性等各方面因素,本课题最终采用PAMAM G5为高分子载体。
选定了基础载体高分子后,本文第二章先采用经典脑靶向头基转铁蛋白(Tf)通过PEG修饰PAMAM,构建了PAMAM-PEG-Tf。UV-vis、~1H-NMR和SDS-PAGE结果显示载体高分子PAMAM-PEG-Tf合成成功。荧光显微镜和流式细胞仪检测细胞内摄取纳米粒的结果表明,PAMAM-PEG-Tf的细胞摄取效率在较低浓度范围内呈浓度依赖性,在高浓度时有饱和趋势。并且,~(125)I放射性标记PAMAM后各载体高分子的体内分布结果显示,经Tf修饰后的载体高分子在脑组织的分布量约为单纯PAMAM的2.25倍。制备的PAMAM-PEG-Tf/DNA纳米粒,其粒径在200 nm左右,zeta电位在13 mV左右,电镜观察显示纳米粒呈球形,表面圆整光滑,具有良好的稳定性。经Tf修饰后,纳米粒在BCECs和脑内的表达均显著高于未修饰的纳米粒。但是,Tf作为脑靶向头基存在一定局限性,如Tf受体介导的双向跨BBB转运功能可能降低通过Tf受体介导入脑的纳米粒在脑内的浓集量、生理状态下BBB上大部分Tf受体可能被内源性Tf占据而降低纳米粒脑靶向性等,需要寻找新的高效的脑靶向头基用于基因递释系统的构建,提高载基因纳米粒的脑靶向性和脑内基因表达效率。
根据文献报道,Lf具有优于Tf和抗Tf受体单克隆抗体OX26的脑内转运效率,提示Lf有可能成为更好的脑靶向头基。但目前关于脑部Lf受体的表征主要是在体外BBB模型上,尚无关于脑组织Lf受体表征的报道,本文第三章对原代培养BCECs和小鼠脑组织上的Lf受体进行表征。共聚焦显微镜和结合动力学实验结果显示,Lf与其受体的结合具有饱和性和竞争抑制现象。结合饱和实验结果显示,脑部Lf受体存在高低两个结合位点,低浓度时主要和高结合位点结合,高浓度时才进一步与低结合位点结合。Lf与BCECs表面高低结合位点的Kd值分别为6.77 nM和4815 nM;与脑细胞膜上高低结合位点的Kd值分别为10.61 nM和2228 nM。
综合第三章研究结果和相关文献可见,Lf作为脑靶向头基具有多项优势,包括:Lf受体介导的跨BBB单向转运功能更利于Lf修饰的纳米基因递释系统在脑组织浓集、生理状态下血循环中Lf的低浓度使内源性Lf不会大量占据脑细胞膜上Lf相关受体等。本文第四章首次采用Lf为脑靶向头基,考察Lf修饰的载体高分子PAMAM-PEG-Lf及其载基因纳米粒的体内外性质。荧光显微镜和流式细胞仪检测结果显示,PAMAM-PEG-Lf的细胞摄取效率在一定范围内呈浓度依赖性,在高浓度时有饱和趋势。并且,在相同浓度比较时,PAMAM-PEG-Lf的细胞摄取和脑内分布高于PAMAM-PEG-Tf,而其他组织的分布量显著下降。在此基础上制备了PAMAM-PEG-Lf/DNA纳米粒,其粒径在210 nm左右,zeta电位在25 mV左右,电镜观察显示纳米粒呈球形,表面圆整光滑,同时透射电镜下观察到纳米粒表面的胶体金颗粒,证实了纳米粒表面存在数十个Lf分子。与Tf修饰的纳米粒比较,经Lf修饰后的纳米粒在BCECs和小鼠脑内的表达均显著提高。纳米粒体内基因表达分布结果显示,PAMAM-PEG-Lf/DNA纳米粒的脑内基因表达量约为PAMAM-PEG-Tf/DNA纳米粒的2.3倍,而全身其他器官的基因表达降低,显示了Lf作为脑靶向头基的优越性。
由于Lf首次作为脑靶向头基、载体高分子PAMAM-PEG-Lf为本课题首次合成,国内外未见报道,该载体高分子及其载基因纳米粒的入脑机制尚不清楚,本文第五章对此进行了探讨。细胞摄取抑制实验结果表明,PAMAM-PEG-Lf保留了PAMAM的高分子特性和Lf的配体-受体结合性质,可以经多种途径包括网格蛋白有被小窝和细胞膜穴样内陷依赖性内吞途径以及巨胞饮途径被原代培养BCECs摄取;PAMAM-PEG-Lf载体高分子及其载基因纳米粒主要通过受体介导的穿细胞转运过程跨越BBB,但仍保留了吸附介导的转运机制。另外,经叶绿素铜脑内示踪实验证实PAMAM-PEG-Lf/DNA纳米粒可跨越BBB进入脑组织。
最后,因文献报道帕金森病(PD)患者脑部神经元和微血管上Lf受体表达显著增加,本文第六章以PD为疾病模型,评价了PAMAM-PEG-Lf包载编码人源性胶质细胞源性神经营养因子(GDNF)的治疗基因(hGDNF)的纳米粒在急性、慢性PD模型大鼠上的药效,设计了多次给药方案,并比较了单次、多次静脉给药后的治疗效果,未见文献报道。结果表明,单次给药时,PAMAM-PEG-Lf/hGDNF纳米粒的脑部基因表达最高,且随时间会降低;多次给药可以持续增加纳米粒的脑内基因表达,5次给药的表达量显著高于单次给药或3次给药,证实了多次给药的可行性和有效性。同时,在6-OHDA单侧损毁急性模型上,多次给药组能显著改善阿朴吗啡诱导的PD症状,明显增加纹状体和黑质中多巴胺能神经元的数目,同时还提高了损毁侧多巴胺(DA)及其代谢产物的量,接近未损毁侧的水平,显示出其优越性。在鱼藤酮慢性模型上,多次给予PAMAM-PEG-Lf/hGDNF纳米粒同样显示出优于单次给药的疗效,多次给药能显著改善PD大鼠的行动能力,提高酪氨酸羟化酶(TH)免疫反应性,同时提高纹状体中DA及其代谢产物的水平。以上实验证明,PAMAM-PEG-Lf/DNA纳米粒是一种高效的非病毒载基因系统,多次静脉注射是一种有效可行的非侵袭性给药方式,可持续提高非病毒载体的脑内基因表达。
Gene therapy offers a promising cure for various brain disorders.However, therapeutic genes themselves,viral vectors and unmodified non-viral vectors can not reach the brain following an intravenous administration due to the presence of blood-brain barrier(BBB).Most genes or current vectors must be given via parenchymal injections which are considered to be highly invasive and unable to deliver genes products to global areas of the brain.Non-viral vectors modified with specific proteins might enable widespread expression of exogenous genes throughout the brain following a non-invasive transvascular pathway.The main problem is the low transfection and expression efficiency.Therefore,developing a brain-targeting and efficient non-viral gene vector to enable the global gene expression in the brain is currently one of the focuses in the field of brain gene therapy.
In this study,a novel efficient brain-targeting non-viral nano-scaled gene delivery system was finally constructed with means of pharmaceutics, macromolecular chemistry and biology.Polyamidoamine(PAMAM) was applied as the main macromolecular gene vector.And lactoferrin(Lf),for the first time,was investigated as a brain-targeting ligand in the design of PAMAM-based nanoparticles (NP) to the brain,using polyethyleneglycol(PEG) as a spacer.There are two important attributes of Lf-modified PAMAM-based nanoparticles (PAMAM-PEG-Lf/DNA NP).They are(1) modified with Lf to target the corresponding receptors in the brain to enhance the brain-targeting ability,(2) employing novel cationic macromolecular material,PAMAM,as the main gene vector,with high gene encapsulation ability
The first part compared the in vitro characteristics including gene incorporation efficiency and transfection efficiency in brain capillary endothelial cells(BCECs) of several cationic vectors.The results of agarose gel electrophoresis and PicoGreen assay showed that all the prepared nanoparticles could effectively encapsulate DNA and protect themselves from the displacement of anionic substances and the digestion of DNase I.The results of fluorescent microscopy and luciferase assay indicated that PAMAM G5(the 5~(th) generation of PAMAM) had a reasonable gene expression. Given both the literature and our results,PAMAM G5 could balance the gene transfection efficiency and cytotoxicity,and thereafter was chosen as the main macromolecular vector in the following studies.
Next,transferrin(Tf) was selected as a brain-targeting ligand,conjugated to PAMAM via bifunctional PEG,yielding PAMAM-PEG-Tf.The results of UV-vis, ~1H-NMR and SDS-PAGE demonstrated the successful synthesis of PAMAM-PEG-Tf. This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,PAMAM-PEG-Tf showed 2.25-fold brain uptake compared to PAMAM in vivo.The PAMAM-PEG-Tf/DNA NP has a particle size around 200 nm and zeta potential around 13 mV.It is spherical and has regular shape under the examination of transmission electron microscope.The modification of Tf significantly enhanced the gene expression of PAMAM-PEG-Tf/DNA NP compared to unmodified nanoparticles in both BCECs and mouse brains.However,there exist some shortcomings of Tf as a brain-targeting ligand.For example,the Tf receptor-mediated bidirectional transcytosis of Tf through the BBB might decrease the brain accumulation of Tf-modified nanoparticles,and most of Tf receptors might be occupied by endogenous Tf under physiological conditions so to reduce the brain-targeting ability of Tf-modified nanoparticles.Consequently,it is necessary to find a novel efficient brain-targeting ligand for construction of brain gene delivery systems,then to enhance the brain-targeting ability and brain gene expression efficiency.
Several lines of evidence demonstrated that Lf had higher brain transport than Tf and a mouse monoclonal antibody against rat Tf receptor(OX26),which suggested that Lf might be a potential brain-targeting ligand.Up to now,there has been little information about the characteristics of Lf receptors in the brain tissue.The third part was to characterize the Lf receptors in the BBB and brain tissue of mice.The results from confocal microscopy showed the presence of Lf receptors on the surface of BCECs and the receptor-mediated endocytosis for Lf to enter the cells.The time course studies demonstrated the saturation phenomenon of binding between Lf and its receptors.Saturation binding analyses revealed that Lf receptors exhibited two classes of binding sites in BCECs(high affinity:dissociation constant(K_d)=6.77 nM; low affinity:K_d=4815 nM) and membrane preparations of mouse brains(high affinity:K_d=10.61 nM;low affinity:K_d=2228 nM).
According to the above-mentioned results and the literature,Lf has several advantages when being a brain-targeting ligand.For example,the Lf receptor-mediated unidirectional transcytosis of Lf through the BBB might increase the brain accumulation of Lf-modified nanoparticles,and most of Lf receptors might not be occupied by endogenous Lf under physiological conditions.Therefore,Lf was,for the first time,exploited as a novel brain-targeting ligand here to synthesize PAMAM-PEG-Lf,and the in vitro and in vivo characteristics of the macromolecular vector and its DNA-loaded nanoparticles were evaluated in the fourth part.This vector showed a concentration-dependent manner in low concentration range and exhibited a trend of saturation in higher concentrations through fluorescent microscopy and flow cytometry.Furthermore,at the same concentration, PAMAM-PEG-Lf showed higher cellular uptake,higher brain accumulation and lower distribution in peripheral tissues,compared to PAMAM-PEG-Tf.The PAMAM-PEG-Lf/DNA NP has a particle size around 210 nm and zeta potential around 25 mV.It is spherical and has regular shape under the examination of transmission electron microscope.Moreover,the observation of colloidal gold on the surface of PAMAM-PEG-Lf/DNA NP demonstrated the successful conjugation of several Lf on the nanoparticle.The modification of Lf significantly enhanced the gene expression of PAMAM-PEG-Lf/DNA NP compared to Tf-modified nanoparticles in both BCECs and mouse brains.The results of quantitative examination of luciferase showed that the brain gene expression of PAMAM-PEG-Lf/DNA NP was about 2.3-fold than that of PAMAM-PEG-Tf/DNA NP,when the gene expression in peripheral tissues markedly reduced.All the results provide evidence that Lf is a promising ligand for the design of gene delivery systems targeting to the brain.
Because Lf was first exploited as a brain-targeting ligand and PAMAM-PEG-Lf was first synthesized in this study,the brain entry mechanisms of this vector and its DNA-loaded nanoparticles remain unclear.These researches were carried out in the fifth part.The results of cellular inhibition showed that PAMAM-PEG-Lf maintained the macromolecular features of PAMAM and the ligand-receptor binding attribute of Lf,and could be taken up by BCECs via multiple pathways including clathrin-,caveolae-dependent endocytosis and macropinocytosis.PAMAM-PEG-Lf and its DNA-loaded nanoparticles could cross the BBB mainly through a receptor-mediated manner,and reserved the adsorptive mediated pathway as well. In addition,the observation of copper chlorophyll under analytical transmission electron microscope demonstrated that PAMAM-PEG-Lf/DNA NP could cross the BBB and reach the brain parenchyma successfully.
As known,an overexpression of Lf receptors was observed in both microvessels and neurons of the substantia nigra in patients with Parkinson's disease(PD).The acute and chronic rat PD models were chosen in the sixth part to evaluate the pharmacodynamics of Lf-modified nanoparticles encapsulating human glial cell line-derived neurotrophic factor gene(hGDNF).And a multiple dosing regimen was for the first time designed to compare the therapeutic effects of single and multiple dosing administrations.The results of single injection showed that PAMAM-PEG-Lf/hGDNF NP had higher brain gene expression compared to other types of nanoparticles,and this gene expression decreased with time.Multiple dosing administrations could enhance and maintain the brain gene products in a higher level.The gene expression of five dosing administrations was significantly higher that that of three dosing administrations.These results demonstrated the feasibility and effectiveness of multiple dosing administrations.In the 6-hydroxydopamine(6-OHDA)-lesioned acute PD model,multiple dosing administrations of PAMAM-PEG-Lf/hGDNF NP could markedly reduce the apomorphine-induced rotations,significantly enhance the number of dopaminergic neurons in striatum and substantia nigra,and increase the content of dopamine(DA) and its metabolites in the lesioned side of rats.In the rotenone chronic PD model, multiple dosing administrations also exhibited better therapeutic effects than single injection.Extending the administration period of PAMAM-PEG-Lf/hGDNF NP could significantly improve locomotor activity,reduce dopaminergic neuronal loss and enhance monoamine neurotransmitter levels on PD rats,which indicating more powerful neuroprotective effects of multiple dosing administrations of Lf-modifined nanoparticles.All the results clearly demonstrate that PAMAM-PEG-Lf/DNA NP is an efficient non-viral gene delivery system,and multiple dosing intravenous administrations could be used as a practical non-invasive means to enhance and maintain the higher brain gene expression of non-viral gene vectors.
引文
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