CN109157530B - 一种包载bdnf基因质粒的peg-plga纳米微球及其制备方法和应用 - Google Patents
一种包载bdnf基因质粒的peg-plga纳米微球及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种包载BDNF基因质粒的PEG‑PLGA纳米微球及其制备方法和应用,该纳米微球由聚乙二醇‑聚乳酸‑羟基乙酸共聚物(PEG‑PLGA)纳米粒包载BDNF基因质粒后得到的的纳米微球。本发明的纳米微球其粒径分布均一,分散性好,具备良好的转染神经干细胞的能力,转染神经干细胞后可高表达脑源性神经营养因子(BDNF)功能蛋白,可用于神经细胞功能保护和损伤修复,可以作为神经干细胞的转染材料在医学领域发挥重要作用,应用前景广阔。
Description
技术领域
本发明属于材料生物学及神经科学技术领域,具体涉及一种包载BDNF基因质粒的PEG-PLGA纳米微球及其制备方法和应用。
背景技术
神经干细胞近年来被证明具有穿血脑屏障能力和趋向迁移至病理部位的潜能,可以在作为种子细胞进行细胞移植治疗的同时作为载体运送基因药物发挥基因治疗的作用。而目前对神经干细胞的基因载体转运系统的研究重点从病毒载体系统转移到了非病毒载体系统。其中研究最多的为脂质体,但是脂质体对神经干细胞的基因转移效率不高,性质不稳定、容易被细胞内外的水解酶消化,成本较高,并具有一定的细胞毒性限制了其应用。
纳米颗粒基因转运体作为近年发展起来的一种新型非病毒基因转运载体受到了越来越多学者的重视。PEG-PLGA作为可生物降解的生物材料之一,由于其可控的缓慢释放,效用确切性,无毒性、无致敏性、无致畸致癌作用及相对廉价被广泛采用。PEG-PLGA纳米基因由于粒径小,通过胞吞方式进入细胞,不需要转染试剂,其自身具有转染能力,且利用PEG-PLGA纳米颗粒转染外源性NSCs能发展更多的功能选项,如多种基因同时表达标记等,是非常理想的转染材料。PEG-PLGA与传统的PLGA相比改善了亲水性和隐身特性,具有低疏水性的药物成分(API)能被有效地包封在PEG-PLGA纳米颗粒中。BDNF主要由神经元产生,在动物模型上的应用表明,它能够防止神经元萎缩、促进轴突生长及促进神经元间新突触形成和神经通路的再生并抑制神经元凋亡,在中枢神经系统受损伤后可以通过发挥神经保护作用、增强神经可塑性、促进神经再生来修复神经组织。
发明内容
发明目的:针对现有技术存在的问题,本发明提供一种包载BDNF基因质粒的PEG-PLGA纳米微球。本发明的纳米微球其粒径分布均一,分散性好,具备良好的转染神经干细胞的能力,转染神经干细胞后可高表达脑源性神经营养因子(BDNF)功能蛋白,可用于神经细胞功能保护和损伤修复,可以作为神经干细胞的转染材料在医学领域发挥重要作用,应用前景广阔。
技术方案:为了实现上述目的,如本发明所述一种包载BDNF基因质粒的PEG-PLGA纳米微球,其特征在于,由聚乙二醇-聚乳酸-羟基乙酸共聚物(PEG-PLGA)纳米粒包载BDNF基因质粒后得到的的纳米微球。
其中,所述聚乙二醇-聚乳酸-羟基乙酸共聚物纳米粒的粒径为217.60±5.40nm。优选为1mgPEG-PLGA纳米粒载有质粒为平均值1.38μg。
其中,所述聚乙二醇-聚乳酸-羟基乙酸共聚物纳米粒的PDI为0.071±0.036。优选使用平均粒径217.60nm的PEG-PLGA包裹BDNF基因质粒,粒径分布均一,单分散性好,少有相互融合现象,PDI优选为平均值0.071,粒径分布集中。
其中,所述包载BDNF基因质粒的PEG-PLGA纳米微球的包封率为(83±3)%,优选包封率为平均值83%。
本发明所述的包载BDNF基因质粒的PEG-PLGA纳米微球的制备方法,包括如下步骤:
(1)将载体材料聚乙二醇-聚乳酸-羟基乙酸共聚物溶于二氯甲烷,将BDNF基因质粒溶液加入该载体材料溶液中,冰浴超声,制备得溶液油包水初乳溶液;
(2)向初乳溶液中加入聚乙烯醇水溶液,冰浴超声,得到水包油包水复乳溶液;
(3)将乳溶液倒入聚乙烯醇(PVA)水溶液中,室温下搅拌,蒸干二氯甲烷,离心洗涤,滤菌,冻干得到包载BDNF基因质粒的PEG-PLGA纳米微球。
作为优选,步骤(1)所述共聚物中聚乙二醇的重均分子量为2000Da,聚乳酸羟基乙酸链段的重均分子量为10000-11500Da
作为优选,步骤(1)所述共聚物中聚乳酸和羟基乙酸两种单体的摩尔比为50:50。即所述聚乳酸羟基乙酸链段由50%乳酸和50%羟基乙酸组成(即为PLGA50/50)。
作为优选,步骤(1)所述BDNF基因质粒溶液与聚乙二醇-聚乳酸-羟基乙酸共聚物的质量比为1:1。
作为优选,步骤(1)中超声处理条件为:用超声细胞破碎仪在80-100W功率下冰浴超声30s,以形成初乳。
作为优选,步骤(2)所述聚乙烯醇水溶液中聚乙烯醇的质量百分比为5%,步骤(3)所述聚乙烯醇水溶液中聚乙烯醇的质量百分比为0.5%。该浓度为聚乙烯醇的最低乳化有效浓度,低于该浓度,乳化效果不足容易导致微球粘连,成型性差。浓度过高则容易导致药物泄漏,载药量降低。
进一步地,步骤(3)所述冻干为冷冻干燥。
本发明所述的包载BDNF基因质粒的PEG-PLGA纳米微球在制备转染神经干细胞并能表达具有脑保护功能的BDNF功能蛋白的基因制剂中的应用。
BDNF基因质粒可来源于质粒扩增,本发明从优宝生物购买小样本后用DH5α细菌扩增然后提纯质粒得来的。扩增后对BDNF基因进行测序。
BDNF基因:
AGGGGCGGGGTTTAGTGGCGAGGACAATAGCAGAGCTGGTTTAGTGAACCGTCAGATCCG
CTAGCGCTACCGGACTCAGATCTGCCACCATGACCATCCTTTTCCTTACTATGGTTATTT
CATACTTTGGTTGCATGAAGGCTGCCCCCATGAAAGAAGCAAACATCCGAGGACAAGGTG
GCTTGGCCTACCCAGGTGTGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAAGG
CAGGTTCAAGAGGCTTGACATCATTGGCTGACACTTTCGAACACGTGATAGAAGAGCTGT
TGGATGAGGACCAGAAAGTTCGGCCCAATGAAGAAAACAATAAGGACGCAGACTTGTACA
CGTCCAGGGTGATGCTCAGTAGTCAAGTGCCTTTGGAGCCTCCTCTTCTCTTTCTGCTGG
AGGAATACAAAAATTACCTAGACGCTGCAAACATGTCCATGAGGGTCCGGCGCCACTCTG
ACCCTGCCCGCCGAGGGGAGCTGAGCGTGTGTGACAGTATTAGTGAGTGGGTAACGGCGG
CAGACAAAAAGACTGCAGTGGACATGTCGGGCGGGACGGTCACAGTCCTTGAAAAGGTCC
CTGTATCAAAAGGCCAACTGAAGCAATACTTCTACGAGACCAAGTGCAATCCCATGGGTT
ACACAAAAGAAGGCTGCAGGGGCATAGACAAAAGGCATTGGAACTCCCAGTGCCGAACTA
CCCAGTCGTACGTGCGGGCCCTTACCATGGATAGCAAAAAGAGAATTGGCTGGCGATTCA
TAAGGATAGACACTTCTTGTGTATGTACATTGACCATTAAAAAGGGGAAGATAGGAATTC
TGCAGTCGACGGTACCGCGGGCCCGGGATCCGCCCCTCTCCCTCCCCCCCCCCTAACGTT
ACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACC
ATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGGAAACCTGTCCTGTCTTCTTGACGAGC
ATTCCTAGGGGTCTTTCCTCTCTCGCAAAGGAATGCAAGTCTGGTGGATGTCGTGAGGAG
CAGTCTCTGGAGCTTCTTGAGACAACCACGTCTGTACGACCCTTGCAGGCAACGATCCCT
CACTGGCGACAGTGACTCTGCGCCAAGCACGTGAAGATACCTGCAAGCGCATCGATTGCA
CGGTGTGAATGGAATACTTGTGA
本发明利用PEG-PLGA包裹BDNF基因质粒(PEG-PLGA购自Sigma-Aldrich公司,BDNF购自优宝生物),制备成新型神经干细胞基因转染材料,探讨其转染外源性神经干细胞后的生物学特性,发展一种新的安全有效的神经干细胞转染方法,以满足未来神经干细胞基础研究和临床转化的需求。
有益效果:与现有技术相比本发明具有如下优点:
本发明的包载BDNF基因质粒的PEG-PLGA纳米微球,采用生物相容性好,可降解的载体材料聚乙二醇-聚乳酸-羟基乙酸共聚物制备的纳米微球。
1.本发明首次采用PEG-PLGA纳米微球作为BDNF基因质粒载体,制备成可以转染神经干细胞并能表达具有脑保护功能的BDNF功能蛋白的基因制剂。
2.本发明制备的纳米微球粒径小,平均粒径217.60nm,表面光滑,外形呈均匀球形,单分散性好。
3.本发明制备的纳米微球包封率好,载药量高,生物相容性好,可以有效转染神经干细胞,且不影响神经干细胞的干性。
附图说明
图1中A和B为PEG-PLGA纳米微球透射电镜图;C和D为PEG-PLGA纳米微球扫描电镜图;
图2为CCK-8法检测两组的神经干细胞(NSCs)增殖活力的示意图;
图3中A为3组神经干细胞表达BDNF蛋白的WB结果;B为WB结果分析;
图4为BDNF基因的RT-PCR结果示意图。
具体实施方式
以下结合附图和实施例对本发明作进一步说明。
实施例1
包载BDNF基因质粒的PEG-PLGA纳米微球的制备
(1)称取200mg聚乙二醇修饰的聚乳酸/羟基乙酸共聚物(PEG-PLGA,聚乙二醇的重均分子量为2000Da,聚乳酸羟基乙酸链段的重均分子量为11500Da或者可以采用重均分子量为10000Da的,聚乳酸和羟基乙酸两种单体的摩尔比为50:50)溶于2ml二氯甲烷(DCM,10%wt/vol)中作为有机油相,将200μlBDNF基因质粒溶液(质粒浓度1000ng/μl)作为水相逐滴加入有机相中,即PEG-PLGA与BDNF基因质粒溶液的质量比为1:1,用超声细胞破碎仪在100W超声功率下冰浴超声30s,制备得溶液初乳(W/O);将初乳逐滴加入5mL 5%聚乙烯醇(PVA)的外水相溶液中剪切,同样功率冰浴超声波降解30s,形成W/O/W复乳溶液;
(2)将复乳倒入20mL 0.5%PVA水溶液中,室温下减压旋转蒸发或磁力搅拌,蒸干二氯甲烷,离心3000r/s,10min,取上清,重复离心一次;离心7000r/s,10min,除去上清液,5ml PBS重悬,450nm滤网滤菌,离心7000r/s,除去上清液,冷冻干燥得到包载BDNF基因质粒的PEG-PLGA纳米微球。
实施例2
对实施例1制备包载BDNF基因质粒的PEG-PLGA纳米微球的理化性质进行以下检测:
(1)扫描电镜和透射电镜观察纳米粒物理形貌
用透射电镜和扫描电镜观察纳米微球形态。
结果如图1所示,扫描电镜和透射电镜下观察包载有BDNF质粒的PEG-PLGA纳米微球可以发现微球外观成球形,表面光滑完整,大小分布均一,单分散性好,少有相互融合现象,未见明显破裂。
(2)马尔文ZS90粒度分析仪测量纳米微球的粒径及Zeta电位
结果示携带质粒的纳米微球粒径为(217.60±5.40)nm,最佳粒径为平均粒径217.60nm,最能体现所制作微球的实际大小,能满足我们对微球用于神经干细胞转染的需求,zeta电位为(-26.33±0.24)mV,最佳电位为平均电位26.33mV;包被PBS的纳米微球粒径为(216.70±5.30)nm,最佳粒径为平均粒径216.70nm,最能体现上述工艺制作的用来满足实际应用要求的微球的实际大小,zeta电位为(-27.23±0.74)mV,最佳电位为平均电位-27.23mV,装载质粒后,PLGA纳米粒呈现出负电荷性质,两者粒径之差为0.9nm,PDI为(0.071±0.036)和(0.081±0.053),最佳PDI为平均PDI,分别为0.071与0.081。
(3)测定PEG-PLGA包裹BDNF基因质粒的包封率及质粒载量
精确称取3个批次各2.5mg冻干后的包载BDNF基因质粒的PEG-PLGA纳米微球(PEG-PLGA-BDNF-NPs)粉末于1.5mlEP管中,加入500μl三氯甲烷使之完全溶解,再加入500μlTE溶液,室温下震荡30min,12000rpm离心5分钟,取上清,紫外分光光度计测定上清中DNA浓度;根据公式计算BDNF纳米微球中质粒含量及包封率。
通过检测上清中质粒的量,计算纳米微球质粒的包封率。得到PEG-PLGA纳米微球的包封率为(83±3)%(n=6)。优选包封率为平均包封率83%,1mgPLGA纳米粒载有质粒(1.38±0.02)μg,最佳载药量为平均载药量:1mgPLGA载有质粒1.38μg,能较好的满足微球用于细胞转染所需的包封率和载药量。
实施例3
包载BDNF基因质粒的PEG-PLGA纳米微球的生物学特性检测
对实施例1制备包载BDNF基因质粒的PEG-PLGA纳米微球的生物学特性进行以下检测:
(1)包载BDNF基因质粒的PEG-PLGA纳米微球对神经干细胞细胞形态和增殖的影响
取体外传代的NSCs,种植到12孔板内,孵育4h后取出,加入含包载BDNF基因质粒的PEG-PLGA纳米微球6mg/ml的完全培养基300μl,37℃,5%CO2孵育3d后用倒置显微镜观察。
取第3代的NSCs,消化为单个细胞后,用培养基将其调整为200000个/ml,加入96孔板,每板设27个实验孔,分为3组,每孔50μl(约10000个细胞);孵育4h后取出,第1组加入含BDNF纳米微球(6mg/ml)的完全培养基50μl,第2组加入含空载纳米微球(6mg/ml)的完全培养基50μl,第3组加入完全培养基50μl,放入培养箱孵育,第1、3、5天取出,每次每组取3个复孔加入10μlCCK-8,设A1孔为空白对照,用于调零,放入培养箱孵育4h后取出,450nm波长处测出各孔吸光度,吸光度大小间接反应纳米微球对NSCs的增殖效应。
结果显示包载BDNF基因质粒的PEG-PLGA纳米微球与NSCs共培养3d后用倒置显微镜观察显示NSCs成球生长,无明显神经球形态改变及分化现象。CCK-8法检测BDNF-NPs、PBS-NPs对细胞增殖影响,结果显示BDNF-NPs对NSCs在第1d、3d均为促进增殖,在第5d为抑制增殖,且相对增值率大于80%;PBS-NPs在第3d为轻度抑制增殖,在第5d显示为明显抑制增殖。见图2,说明转染用浓度的纳米微球有满意的细胞相容性,对神经干细胞生物活性影响为正面作用。
(2)携载BDNF基因质粒纳米微球的外源性NSCs的分泌能力检测
细胞分泌BDNF蛋白用Western-Blot法测定。用Rippa裂解液提取按上述方法培养第四天的细胞总蛋白,包括转染组、PBS载体组、空白对照组。取蛋白样品10μl用于蛋白定量,剩余样品按5:1加入SDS buffer,沸水浴5min。每孔加样蛋白约20μg,行10%SDS聚丙烯酰胺凝胶电泳,采用半干法将蛋白转移至NC膜。用5%脱脂奶封闭,加入BDNF多克隆抗体(1:5000)和β-actin多克隆抗体(1:5000)过夜后PBS洗涤3遍,二抗为1:5000稀释的辣根过氧化酶标记的山羊抗兔抗体和山羊抗小鼠抗体,室温孵育60min,PBS洗涤后用BCIP-NBT液于室温下避光显色,扫描图像,分析结果,以β-actin作为内参照。
BDNF mRNA采用定量RT-PCR法测定。用Trizol试剂盒(OMEGA total RNA kit)提取RNA,逆转录为cDNA,用于RT-PCR,进行定量分析。
Western-Blot结果显示,载BDNF基因PEG-PLGA-NPs共培养组在28kDa左右处可见一清晰条带,灰度明显高于空PEG-PLGA载体组和空白对照组,说明BDNF基因片段成功转染入神经干细胞,并能表达具有生物活性的BDNF蛋白,见图3。
定量RT-PCR结果显示载质粒PEG-PLGA转染组细胞BDNF基因含量明显高于空PEG-PLGA载体组和空白对照组,说明外源性BDNF mRNA在转染细胞中得到表达,见图4。
序列表
<110> 东南大学
<120> 一种包载BDNF基因质粒的PEG-PLGA纳米微球及其制备方法和应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1223
<212> DNA
<213> BDNF基因质粒(BDNF)
<400> 1
aggggcgggg tttagtggcg aggacaatag cagagctggt ttagtgaacc gtcagatccg 60
ctagcgctac cggactcaga tctgccacca tgaccatcct tttccttact atggttattt 120
catactttgg ttgcatgaag gctgccccca tgaaagaagc aaacatccga ggacaaggtg 180
gcttggccta cccaggtgtg cggacccatg ggactctgga gagcgtgaat gggcccaagg 240
caggttcaag aggcttgaca tcattggctg acactttcga acacgtgata gaagagctgt 300
tggatgagga ccagaaagtt cggcccaatg aagaaaacaa taaggacgca gacttgtaca 360
cgtccagggt gatgctcagt agtcaagtgc ctttggagcc tcctcttctc tttctgctgg 420
aggaatacaa aaattaccta gacgctgcaa acatgtccat gagggtccgg cgccactctg 480
accctgcccg ccgaggggag ctgagcgtgt gtgacagtat tagtgagtgg gtaacggcgg 540
cagacaaaaa gactgcagtg gacatgtcgg gcgggacggt cacagtcctt gaaaaggtcc 600
ctgtatcaaa aggccaactg aagcaatact tctacgagac caagtgcaat cccatgggtt 660
acacaaaaga aggctgcagg ggcatagaca aaaggcattg gaactcccag tgccgaacta 720
cccagtcgta cgtgcgggcc cttaccatgg atagcaaaaa gagaattggc tggcgattca 780
taaggataga cacttcttgt gtatgtacat tgaccattaa aaaggggaag ataggaattc 840
tgcagtcgac ggtaccgcgg gcccgggatc cgcccctctc cctccccccc ccctaacgtt 900
actggccgaa gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc 960
atattgccgt cttttggcaa tgtgagggcc cgggaaacct gtcctgtctt cttgacgagc 1020
attcctaggg gtctttcctc tctcgcaaag gaatgcaagt ctggtggatg tcgtgaggag 1080
cagtctctgg agcttcttga gacaaccacg tctgtacgac ccttgcaggc aacgatccct 1140
cactggcgac agtgactctg cgccaagcac gtgaagatac ctgcaagcgc atcgattgca 1200
cggtgtgaat ggaatacttg tga 1223
Claims (10)
1.一种包载BDNF基因质粒的PEG-PLGA纳米微球,其特征在于,由聚乙二醇-聚乳酸-羟基乙酸共聚物PEG-PLGA纳米粒包载BDNF基因质粒后得到的纳米微球;所述的包载BDNF基因质粒的PEG-PLGA纳米微球的制备方法,包括如下步骤:
(1)将载体材料聚乙二醇-聚乳酸-羟基乙酸共聚物溶于二氯甲烷,将BDNF基因质粒溶液加入该载体材料溶液中,冰浴超声,制备得溶液油包水初乳溶液;
(2)向初乳溶液中加入聚乙烯醇水溶液,冰浴超声,得到水包油包水复乳溶液;
(3)将乳溶液倒入聚乙烯醇水溶液中,室温下搅拌,蒸干二氯甲烷,离心洗涤,滤菌,冻干得到包载BDNF基因质粒的PEG-PLGA纳米微球;
步骤(1)所述BDNF基因质粒溶液与聚乙二醇-聚乳酸-羟基乙酸共聚物的质量比为1:1;步骤(2)所述聚乙烯醇水溶液的质量百分比为5%,步骤(3)所述聚乙烯醇水溶液的质量百分比为0.5%。
2.根据权利要求1所述的包载BDNF基因质粒的PEG-PLGA纳米微球,其特征在于,所述聚乙二醇-聚乳酸-羟基乙酸共聚物纳米粒的粒径为217.60±5.40nm。
3.根据权利要求1所述的包载BDNF基因质粒的PEG-PLGA纳米微球,其特征在于,所述聚乙二醇-聚乳酸-羟基乙酸共聚物纳米粒的PDI为0.071±0.036。
4.根据权利要求1所述的包载BDNF基因质粒的PEG-PLGA纳米微球,其特征在于,所述包载BDNF基因质粒的PEG-PLGA纳米微球的包封率为(83±3)%。
5.一种权利要求1所述的包载BDNF基因质粒的PEG-PLGA纳米微球的制备方法,其特征在于,包括如下步骤:
(1)将载体材料聚乙二醇-聚乳酸-羟基乙酸共聚物溶于二氯甲烷,将BDNF基因质粒溶液加入该载体材料溶液中,冰浴超声,制备得溶液油包水初乳溶液;
(2)向初乳溶液中加入聚乙烯醇水溶液,冰浴超声,得到水包油包水复乳溶液;
(3)将乳溶液倒入聚乙烯醇水溶液中,室温下搅拌,蒸干二氯甲烷,离心洗涤,滤菌,冻干得到包载BDNF基因质粒的PEG-PLGA纳米微球。
6.根据权利要求5所述的制备方法,其特征在于,步骤(1)所述共聚物中聚乙二醇的重均分子量为2000Da,聚乳酸羟基乙酸链段的重均分子量为10000-11500Da。
7.根据权利要求5所述的制备方法,其特征在于,步骤(1)所述共聚物中聚乳酸和羟基乙酸两种单体的摩尔比为50:50。
8.根据权利要求5所述的制备方法,其特征在于,步骤(1)所述BDNF基因质粒溶液与聚乙二醇-聚乳酸-羟基乙酸共聚物的质量比为1:1。
9.根据权利要求5所述的制备方法,其特征在于,步骤(2)所述聚乙烯醇水溶液的质量百分比为5%,步骤(3)所述聚乙烯醇水溶液的质量百分比为0.5%。
10.一种权利要求1所述的包载BDNF基因质粒的PEG-PLGA纳米微球在制备与转染神经干细胞并能表达具有脑保护功能的BDNF功能蛋白的基因制剂中的应用。
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