CN111388677B - 药物载体、基于crispr基因编辑技术的脑靶向纳米药物及其制备方法和应用 - Google Patents

药物载体、基于crispr基因编辑技术的脑靶向纳米药物及其制备方法和应用 Download PDF

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CN111388677B
CN111388677B CN202010214295.8A CN202010214295A CN111388677B CN 111388677 B CN111388677 B CN 111388677B CN 202010214295 A CN202010214295 A CN 202010214295A CN 111388677 B CN111388677 B CN 111388677B
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drug
cas9
nano
sgrna
brain
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CN111388677A (zh
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阮卫民
焦明珠
师冰洋
郑蒙
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Henan University
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Abstract

本发明提供一种药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其制备方法和应用。本发明中提供的纳米药物，含有由Cas9/sgRNA和药物载体偶联制得纳米粒子，Cas9/sgRNA切割效率高，药物载体中Angiopep多肽有助于穿过血脑屏障(BBB)及靶向至脑肿瘤细胞，PEG可以有效地延长血液循环周期且具有很好的生物相容性，胍基Gu+不仅可以通过静电作用还可以形成盐桥及氢键作用与核糖蛋白复合体结合，少量的氟可以增强纳米药物的稳定性，大大延长纳米药物药代动力学半衰期。使用该药物载体可将治疗药物有效运输到病灶部位。该纳米药物可在基因水平上进行肿瘤抑制及治疗。

Description

药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其 制备方法和应用
技术领域
本发明涉及纳米药物领域，尤其涉及一种药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其制备方法和应用。
背景技术
脑胶质瘤是原发性颅脑恶性肿瘤。其发病率占颅内肿瘤的35.2～
61.0％，具有发病率高、复发率高、死亡率高、治愈率低等特点。目前用于治疗胶质瘤的临床手段，主要包括手术、放疗、化疗等手段。
血脑屏障(blood brain barrier,BBB)使得人脑胶质瘤成为癌症治疗中最棘手的肿瘤之一。BBB为脑补的自我平衡防御机制，一方面，它保证中枢神经系统免受外来物质侵扰，维持高效的稳态，同时为脑内输入营养物质；另一方面，BBB的致密结构也阻碍了治疗药物通过非入侵性给药进入脑内。因此，发掘研究能协助纳米药物突破BBB的药物或靶向分子是治疗脑部疾病的当务之急。
发明内容
本发明的目的在于提供一种药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其制备方法和应用。
为实现以上目的，本发明提供以下技术方案。
本发明第一方面提供一种药物载体，包括聚合物mPEG-P(GPMA,FPMA)和聚合物Ang-PEG-PGPMA；
所述mPEG-P(GPMA,FPMA)的结构式为：
Figure BDA0002423877210000021
所述聚合物Ang-PEG-PGPMA的结构式为:
Figure BDA0002423877210000022
其中，n为35～45，x1为15～20，y为2～4，m为75～85，x2＝x1。
作为上述技术方案的进一步改进，聚合物mPEG-P(GPMA,FPMA)为mPEG2K-P(GPMA3K-4K,FPMA0.5K-1K)。
作为上述技术方案的进一步改进，聚合物Ang-PEG-PGPMA为Ang-PEG3.4K-PGPMA4K。
本发明第二方面提供一种基于CRISPR基因编辑技术的脑靶向纳米药物，包括由治疗药物与第一方面所述的药物载体偶联得到的纳米粒子。
作为上述技术方案的进一步改进，所述治疗药物为Cas9/sgRNA，所述Cas9/sgRNA由Cas9蛋白与sgRNA复合得到。
作为上述技术方案的进一步改进，所述sgRNA为靶向致癌基因PLK1的sgRNA。
作为上述技术方案的进一步改进，所述靶向致癌基因PLK1的sgRNA的序列如SEQID NO：1所示。
作为上述技术方案的进一步改进，以摩尔份数计，所述Cas9/sgRNA中，Cas9蛋白与sgRNA的比例为1:(1～1.5)。
作为上述技术方案的进一步改进，以摩尔份数计，所述纳米粒子中含有：1份Cas9/sgRNA、1～3份mPEG-P(GPMA,FPMA)和4～12份Ang-PEG-PGPMA。
本发明第三方面提供第二方面所述的基于CRISPR基因编辑技术的脑靶向纳米药物的制备方法，包括：
步骤a.将聚合物mPEG-P(GPMA,FPMA)、聚合物Ang-PEG-PGPMA溶解于缓冲溶液得到混合溶液A；
步骤b.在混合溶液A中加入Cas9/sgRNA，反应得到纳米粒子Ang-NP@Cas9/sgRNA。
作为上述技术方案的进一步改进，所述缓冲溶液选自HEPES缓冲溶液、PBS缓冲液中的任意一种。
作为上述技术方案的进一步改进，所述缓冲溶液的摩尔浓度为10～50mM。
作为上述技术方案的进一步改进，所述缓冲溶液的pH为7.4～8.0。
作为上述技术方案的进一步改进，步骤b中反应的条件为混匀室温静置。
作为上述技术方案的进一步改进，静置时间为30～120min。
本发明第四方面提供第二方面所述的基于CRISPR基因编辑技术的脑靶向纳米药物在制备肿瘤治疗药物中的应用。
作为上述技术方案的进一步改进，所述肿瘤为脑胶质瘤。
本发明的有益效果：本发明提供的药物载体中Angiopep多肽有助于穿过BBB及靶向至肿瘤细胞，PEG可以有效地延长血液循环周期且具有很好的生物相容性，胍基Gu+不仅可以通过静电作用还可以形成盐桥及氢键作用与核糖蛋白复合体结合，少量的氟可以增强纳米药物的稳定性，大大延长纳米药物药代动力学半衰期。使用该药物载体可将治疗药物有效运输到病灶部位。
本发明中提供的基于CRISPR基因编辑技术的脑靶向纳米药物，含有由Cas9/sgRNA和药物载体偶联制得纳米粒子，Cas9/sgRNA切割效率高，药物载体中Angiopep多肽有助于穿过血脑屏障(BBB)及靶向至脑肿瘤细胞，PEG可以有效地延长血液循环周期且具有很好的生物相容性，胍基Gu+不仅可以通过静电作用还可以形成盐桥及氢键作用与核糖蛋白复合体结合，少量的氟可以增强纳米药物的稳定性，大大延长纳米药物药代动力学半衰期。使用该药物载体可将治疗药物有效运输到病灶部位。该纳米药物可在基因水平上进行肿瘤抑制及治疗。
附图说明
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对本发明范围的限定。
图1为1H-NMR图谱，其中图1中(a)为GPMA单体的1H-NMR图谱，图1中(b)为mPEG-CPADN的1H-NMR图谱，图1中(c)为Mal-PEG-CPADN的1H-NMR图谱，图1中(d)为mPEG-P(GPMA-FPMA)的1H-NMR图谱。
图2为纳米药物表征结果，其中，图2中(a)为纳米药物的粒径图，图2中(b)为纳米药物的TEM图像，图2中(c)为圆二色光谱检测结果。
图3为纳米药物细胞水平实验结果，其中，图3中(a)为流式细胞检测结果，图3中(b)为共聚焦实验结果，图3中(c)为MTT检测结果，图3中(d)为PLK1基因敲除结果，图3中(e)为PLK1基因被敲除后蛋白表达结果。
图4为纳米药物动物水平实验结果，其中，图4中(a)为体内药代动力学研究结果，图4中(b)为纳米药物肿瘤靶向效果，图4中(c)、(d)为纳米药物在小鼠体内各个器官的分布情况。
具体实施方式
如本文所用之术语：
“由……制备”与“包含”同义。本文中所用的术语“包含”、“包括”、“具有”、“含有”或其任何其它变形，意在覆盖非排它性的包括。例如，包含所列要素的组合物、步骤、方法、制品或装置不必仅限于那些要素，而是可以包括未明确列出的其它要素或此种组合物、步骤、方法、制品或装置所固有的要素。
连接词“由……组成”排除任何未指出的要素、步骤或组分。如果用于权利要求中，此短语将使权利要求为封闭式，使其不包含除那些描述的材料以外的材料，但与其相关的常规杂质除外。当短语“由……组成”出现在权利要求主体的子句中而不是紧接在主题之后时，其仅限定在该子句中描述的要素；其它要素并不被排除在作为整体的所述权利要求之外。
当量、浓度、或者其它值或参数以范围、优选范围、或一系列上限优选值和下限优选值限定的范围表示时，这应当被理解为具体公开了由任何范围上限或优选值与任何范围下限或优选值的任一配对所形成的所有范围，而不论该范围是否单独公开了。例如，当公开了范围“1～5”时，所描述的范围应被解释为包括范围“1～4”、“1～3”、“1～2”、“1～2和4～5”、“1～3和5”等。当数值范围在本文中被描述时，除非另外说明，否则该范围意图包括其端值和在该范围内的所有整数和分数。
在这些实施例中，除非另有指明，所述的份和百分比均按质量计。
“质量份”指表示多个组分的质量比例关系的基本计量单位，1份可表示任意的单位质量，如可以表示为1g，也可表示2.689g等。假如我们说A组分的质量份为a份，B组分的质量份为b份，则表示A组分的质量和B组分的质量之比a：b。或者，表示A组分的质量为aK，B组分的质量为bK(K为任意数，表示倍数因子)。不可误解的是，与质量份数不同的是，所有组分的质量份之和并不受限于100份之限制。
“和/或”用于表示所说明的情况的一者或两者均可能发生，例如，A和/或B包括(A和B)和(A或B)。
“GPMA”是指胍基单体，结构式为：
Figure BDA0002423877210000064
“FPMA”是指2,2,3,3-四氟丙基甲基丙烯酸酯，结构式为：
Figure BDA0002423877210000061
“CPADN”是指4-氰基-4-(苯基硫代甲酰硫基)戊酸，结构式为
Figure BDA0002423877210000062
本发明提供一种药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其制备方法和应用，具体地，本发明提供以下技术方案。
本发明第一方面提供一种药物载体，包括聚合物mPEG-P(GPMA,FPMA)和聚合物Ang-PEG-PGPMA；
所述mPEG-P(GPMA,FPMA)的结构式为：
Figure BDA0002423877210000063
所述聚合物Ang-PEG-PGPMA的结构式为:
Figure BDA0002423877210000071
其中，n为35～45，x1为15～20，y为2～4，m为75～85，x2＝x1。
本发明的药物载体中Angiopep多肽有助于穿过BBB及靶向至肿瘤细胞，PEG可以有效地延长血液循环周期且具有很好的生物相容性，胍基Gu+不仅可以通过静电作用还可以形成盐桥及氢键作用与核糖蛋白复合体结合，少量的氟可以增强纳米药物的稳定性，大大延长纳米药物药代动力学半衰期。使用该药物载体可将治疗药物有效运输到病灶部位。
可选地，聚合物mPEG-P(GPMA,FPMA)为mPEG2K-P(GPMA3K-4K,FPMA0.5K-1K)。
可选地，聚合物Ang-PEG-PGPMA为Ang-PEG3.4K-PGPMA4K。
本发明第二方面提供一种基于CRISPR基因编辑技术的脑靶向纳米药物，包括：由治疗药物与第一方面所述的药物载体偶联得到的纳米粒子。
可选地，所述治疗药物为复合物Cas9/sgRNA，所述Cas9/sgRNA由Cas9蛋白与sgRNA复合得到。
可选地，所述sgRNA为靶向致癌基因PLK1的sgRNA。
可选地，所述靶向致癌基因PLK1的sgRNA的序列如SEQ ID NO：1所示。
polo样激酶I(PLK1)是一种丝苏氨酸蛋白激酶,在细胞周期中发挥重要的作用。研究发现,PLK1在脑胶质瘤中的表达明显高于正常细胞,抑制PLK1的表达能显著抑制肿瘤的生长,而对正常细胞没有明显的抑制作用。为此，挑选了基因PLK1进行基因编辑，在基因水平上进行肿瘤抑制及治疗。序列如SEQ ID NO：1所示的sgRNA与Cas9蛋白复合得到的Cas9/sgRNA靶向致癌基因PLK1基因，且对PLK1基因具有高效切割能力。
可选地，以摩尔份数计，所述Cas9/sgRNA中，Cas9蛋白与sgRNA的比例为1:(1～1.5)。
可选地，以摩尔份数计，所述纳米粒子中含有：1份Cas9/sgRNA、1～3份mPEG-P(GPMA,FPMA)和4～12份Ang-PEG-PGPMA。在该比例下，制备得到的纳米粒子粒径较好，使用较少材料装载药物。
本发明第三方面提供第二方面所述的基于CRISPR基因编辑技术的脑靶向纳米药物的制备方法，包括：
步骤a.将聚合物mPEG-P(GPMA,FPMA)、聚合物Ang-PEG-PGPMA溶解于缓冲溶液得到混合溶液A；
步骤b.在混合溶液A中加入Cas9/sgRNA，反应得到纳米粒子Ang-NP@Cas9/sgRNA。
上述制备方法中的原料可自行合成，也可直接购买。本发明中的制备方法反应条件温和，操作简单。
可选地，所述缓冲溶液选自HEPES缓冲溶液、PBS缓冲液中的任意一种。
可选地，所述缓冲溶液的摩尔浓度为10～50mM。
可选地，所述缓冲溶液的pH为7.4～8.0。
可选地，步骤b中反应的条件为混匀室温静置；优选地，静置时间为30～120min。
本发明第四方面提供第二方面所述的基于CRISPR基因编辑技术的脑靶向纳米药物在制备肿瘤治疗药物中的应用；优选地，所述肿瘤为脑胶质瘤。
下面将结合具体实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限制本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
实施例1
纳米药物的制备方法
本实施例中纳米药物为纳米粒子Ang-NP@Cas9/sgRNA，纳米粒子的制备方法包括：体外转录sgRNA、合成高分子聚合物及制备纳米粒子，转录得到的sgRNA的靶序列为TACCTACGGCAAATTGTGCT(SEQ ID NO：1)。具体步骤如下：
1、体外转录sgRNA
体外转录模仿体内转录的环境，在整个操作过程中无RNA酶,目的即防止转录后sgRNA发生降解，样品要进行分装保存在-80℃冰箱中。具体步骤如下：
1.1退火寡糖oligo(总体系20μL)
Figure BDA0002423877210000091
95℃，5min之后放置室温过夜(或者沸水自然冷却至室温)。
1.2加入1μL Taq酶(10X PCR polymeranse)1h,37℃。
1.3 DNA纯化(康为Clean up试剂盒)，纯化后4％琼脂糖凝胶电泳验证，纯化的DNA在-20℃冰箱保存。
1.4体外转录sgRNA
转录体系如下：
Figure BDA0002423877210000092
Figure BDA0002423877210000101
充分混匀，37℃过夜。之后加入10μL DNA Buffer和70μL DEPC水，1μL DNA酶，37℃，1h。
1.5使用碧云天Trizon试剂盒纯化RNA，纯化的样品在-80℃冰箱保存。
2、合成高分子聚合物
2.1合成聚合物mPEG2K-P(GPMA4K,FPMA0.6K)
合成路线如下：
Figure BDA0002423877210000102
具体步骤为：
2.1.1制备GPMA单体
1)16mL DMF中加入APM(1.0g,5.6mmol)，H-pyrazde-carboxamidrine(0.82g,5.6mmol)，对二苯酚(10mg)，加入TEA(1230mg,12.3mmol)后室温搅拌24h。
2)停止反应，将反应液离心(8000rpm,5min),收集上清使用0.22μm的滤头进行过滤将不容物除去。
3)之后加入乙醚(100mL)清洗，离心(8000rpm,5min),留沉淀。
4)使用20mL乙腈，1mL TEA洗两次，留取沉淀。
5)使用二氯甲烷(30mL)洗一次，最后使用乙醚洗一次，真空干燥得到淡黄色透明粘状产物，通氮气-20℃避光保存。
2.1.2合成mPEG2K-CPADN
mPEG-NH2、CPADN以摩尔比1:3反应，mPEG-NH2，CPADN-NHS混合加入到单颈瓶中，室温搅拌10h，0.22μm滤头过滤，冰乙醚沉淀(8000rpm，4℃，5min，洗至上清无色)，每次取3mL二氯甲烷溶解，滴加冰乙醚中沉淀洗涤。
2.1.3合成聚合物mPEG2K-P(GPMA4K,FPMA0.6K)
A.FPMA、GPMA、CPADN的摩尔比为8:30:1。
GPMA(600μmol)，FPMA(140μmol)，PEG-CPADN(20μmol)，AIBN(3μmol)，先将他们各自溶于DMF中，加入反应瓶中，最后加入AIBN，加入AIBN后将油浴锅缓慢升温至65℃，搅拌48h。
B.使用3500D的透析袋使用水进行透析，2h次进行换水48h，将多余未结合的GPMA透析除去，最后使用真空冷冻干燥进行干燥，样品-20℃避光保存。
2.2合成聚合物Ang-PEG3.4K-PGPMA4K(方法同2.1)。
合成路线如下：
Figure BDA0002423877210000121
具体步骤如下：
1)合成Mal-PEG3.4K-CPADN
A.活化CPADN
CPADN、NHS、DCC、TEA以摩尔比1:1:1.2:10的比例反应。
CPADN(0.375mmol，37mg)，NHS(0.4125mmol，15.2mg)，DCC(0.4125mmol，32.6mg)，TEA(3.75mmol，150μL)，NHS溶于1mL DMF中，CPADN溶于10mL二氯甲烷，加三乙胺，DCC缓慢滴加，需要冰浴，待冰缓慢融化为室温撤去，搅拌过夜。
B.加入Mal-PEG-NH2(44mmol，150mg)，室温搅拌10h，0.22μm滤头过滤，冰乙醚沉淀(8000rpm，4℃，5min，洗至上清无色)，每次取3mL二氯甲烷溶解，滴加冰乙醚中沉淀洗涤。
2)合成Ang-PEG3.4K-CPADN
Mal-PEG-CPADN(45mg)，Angipep-SH(50mg)在DMSO中，37℃，搅拌过夜，水透析(3.5K透析袋)48h，冷冻干燥，经BCA检测Angipep接上的量。
3)合成聚合物Ang-PEG3.4K-PGPMA4K(方式同2.1.3)。
GPMA单体、mPEG-CPADN、Mal-PEG-CPADN、mPEG-P(GPMA-FPMA)的1H-NMR图谱如图1所示。
3、制备纳米粒子
3.1Cas9与sgRNA进行结合：Cas9与sgRNA以1:1.2摩尔比在10mM HEPES(pH 7.4)缓冲溶液进行混合，室温静置5min。
3.2制备纳米粒子NP@Cas9/sgRNA：将mPEG2K-P(GPMA4K,FPMA0.6K)溶解在10mM HEPES缓冲溶液，与Cas9/sgRNA以1:2摩尔比混合静置30分钟以形成NP@Cas9/sgRNA，当sgRNA为sgPLK1时，制备得到NP@Cas9/sgPLK1。
3.3制备纳米粒子Ang-NP@Cas9/sgRNA：将mPEG2K-P(GPMA4K,FPMA0.6K)和Ang-PEG3.4K-PGPMA4K以摩尔比1：4混合溶解在10mM HEPES缓冲溶液中，之后与Cas9/sgRNA混匀室温静置30分钟以形成Ang-NP@Cas9/sgRNA，当sgRNA为sgPLK1时，制备得到Ang-NP@Cas9/sgPLK1。
实施例2～4中使用的纳米药物为实施例1中制备的纳米药物。
实施例2
纳米药物表征结果
Ang-NP@Cas9/sgPLK1和Free Cas9/sgPLK1(即Cas9/sgPLK1复合物)的粒径图如图2中(a)所示，纳米药物的粒径及电位如表1所示。图2中(b)为Ang-NP@Cas9/sgPLK1的TEM图像。Cas9/sgPLK1复合物粒径大约在30nm，带有负电荷，被聚合物包裹后，粒径为149nm，稳定性明显增强，均一性很好，表面带有微弱的正电荷，为纳米药物进入体内治疗肿瘤提供可能性。
表1纳米药物的粒径及电位
Figure BDA0002423877210000131
Figure BDA0002423877210000141
图2中(c)为圆二色光谱检测结果，本实施例中以BSA蛋白为例，使用mPEG2K-P(GPMA4K,FPMA0.6K)装载BSA蛋白制备纳米药物(NPs-BSA)，对比Free BSA与NPs-BSA在190～300nm,25℃的条件下峰值的变化。结果证明mPEG2K-P(GPMA4K,FPMA0.6K)结合蛋白后没有破坏蛋白表面结果，这说明蛋白与mPEG2K-P(GPMA4K,FPMA0.6K)结合后可以发挥作用。
实施例3、4通过细胞水平实验和动物水平实验验证了实施例1中制备的纳米药物的效果。
实施例3
细胞水平实验
1、流式细胞分析细胞内吞
在流式细胞仪测试中，将U87-Luc细胞种在6孔细胞培养板内(1×105细胞/孔)在37℃培养贴壁16～24h，加入300μL的标记有荧光的Cas9-Alexa647制备的Free Cas9/sgPLK1,NP@Cas9/sgPLK1,Ang-NP@Cas9/sgPLK1，Ang-NP@Cas9/sgScr(Cas9浓度为10nM)以及PBS培养4h,吸走样品，用200μL胰酶消化细胞，37℃，3min，加入500μL新鲜培养基终止消化。得到的细胞悬浮液在1000×g离心3min，用PBS洗两次，再次分散在500μL PBS，进行流式细胞仪(BD FACS Calibur，Becton Dickinson,USA)测试。其中，其中sgScr指代没有治疗效果的阴性对照。
检测结果如图3中(a)所示，由图3中(a)可知，证明Ang-NP@Cas9/sgPLK1对U87-Luc细胞有很好的靶向效果。
2、激光共聚焦显微镜分析
2.1将U87-Luc细胞铺于含显微镜载玻片的24孔细胞培养板里(5×104细胞/孔)培养24h后，加入300μL的Free Cas9/sgPLK1,NP@Cas9/sgPLK1,Ang-NP@Cas9/sgPLK1(Cas9浓度为50nM)以及PBS孵育4h后进行制样。
2.2固定：PBS洗三遍使用4％多聚甲醛进行固定，37℃，15min后将多聚甲醛吸走，PBS洗三次。
2.3细胞染色
2.3.1鬼笔环肽-FITC：
A、0.1％TRITON X-100渗透15min，PBS洗3次；
B、10μg/ml fluorescent phalloidin染色20～40min，PBS洗3次。
2.3.2Hochest 33342浓度为10μg/ml,染色5～10min。
加的液体体积浸没样品即可，如：24孔板加200μL体积即可。
2.4封片：在载玻片上加少量(～5μL)的抗荧光猝灭剂，盖玻片上有细胞的那一面贴近载玻片，晾至凝固放于共聚焦盒子中避光保存。
共聚焦实验结果如图3中(b)所示，从图3中(b)可知，Ang-NP@Cas9/sgPLK1可以很好进入细胞，并且可以成功进入细胞核，为基因编辑提供可能性。
3、MTT方法检测纳米药物的细胞毒性
MTT检测结果如图3中(c)所示，从图3中(c)可知，Ang-NP@Cas9/sgPLK1对细胞毒性很小，有很好的生物相容性。
4、通过限制性内切酶法检测纳米药物基因编辑效果
4.1转染
1)将U87-Luc细胞铺在24孔板中(5×104细胞/孔)，使细胞贴壁16-24h。
2)加入300μL Free Cas9/sgPLK1，NP@Cas9/sgPLK1，Ang-NP@Cas9/sgPLK1，Ang-NP@Cas9/sgScr(Cas9浓度为50nM)与细胞孵育6h后更换新鲜培养基，继续培养72h后收集细胞。其中sgScr指代没有治疗效果的阴性对照。
4.2提取基因组
收集细胞后使用试剂盒提取细胞基因组。
4.3PCR技术扩增PLK1基因组片段
将目的片段使用高保真酶进行PCR，PCR反应体系如下表所示。
Figure BDA0002423877210000161
4.4酶切验证基因水平编辑效果
使用NEB的BstAP I(R0654S)对PCR片段进行酶切，2％琼脂糖电泳后，凝胶成像仪采集图像，最后使用image J软件灰度分析编辑效率。
PLK1基因敲除结果如图3中(d)所示，由图3中(d)可知，基因水平可以达到32％的效果。
5、通过Western Blot探索Cas9基因编辑后对蛋白表达的影响
5.1转染
1)将U87-Luc细胞铺在6孔板中(1×105细胞/孔)，使细胞贴壁16～24h。
2)加入500μL Free Cas9/sgPLK1,NP@Cas9/sgPLK1,Ang-NP@Cas9/sgPLK1，Ang-NP@Cas9/sgScr(Cas9浓度为50nM)与细胞孵育6h后更换新鲜培养基，继续培养96h后收集细胞。其中sgScr指代没有治疗效果的阴性对照。
5.2提取细胞全蛋白
1)使用含有PMSF的RIPA(强)将蛋白裂解，收集蛋白样品。
5.3跑胶
1)蛋白变性：将样品100℃，5min把蛋白变性。
2)使用10％SDS-PAGE胶进行电泳。
3)转模(300mA,30min)。
4)封闭(5％脱脂牛奶，室温，2h)。
5)孵育一抗(4℃，过夜)。
6)孵育二抗(使用带荧光标记的二抗室温孵育1h)。
5.4显影
结果如图3中(e)所示，Western Blot验证PLK1基因被敲除后对蛋白表达量的降低，最终纳米药物基因编辑后的蛋白表达量可以降低到33％。
实施例4
动物水平实验
1、药代动力学实验
对比Free Cas9/sgPLK1,NP@Cas9/sgPLK1，Ang-NP@Cas9/sgPLK1在小鼠体内药代动力学分析，6～8周BALB/c小鼠随机分组(每组平行3只)，待尾部静脉注射完药物后在3min、5min、10min、15min、20min、30min、45min、60min、1.5h、2h、3h、4h、6h、8h、10h使用毛细吸管眼部取血。
体内药代动力学研究结果如图4中(a)所示，由图4中(a)可知，Ang-NP@Cas9/sgPLK1在体内的循环时间较长，与NP@Cas9/sgPLK1相当(图4中(a))，而长于Free Cas9/sgPLK1，说明Ang-NP@Cas9/sgPLK1具有较长的血液循环时间，延长药物在血液中的循环时间。
2、纳米药物在小鼠体内的穿越BBB及肿瘤靶向效果
U87-Luc脑胶质肿瘤原位模型的建立是在BALB/c裸鼠(18～20g，6～8周龄)脑部移植肿瘤组织。选取Ang-NP@Cas9/sgPLK1，NP@Cas9/sgPLK1，两组对照，每只小鼠注射Cas9的量为15μg，每组3只平行，分别在0h、2h、4h、6h、8h、12h、24h使用小动物成像仪进行荧光照射，观察纳米粒子的荧光强度判断纳米药物肿瘤靶向效果，结果如图4中(b)所示。
从荧光强度可以看出Ang-NP@Cas9/sgPLK1在动物脑部具有较强的荧光强度，说明Ang-NP@Cas9/sgPLK1在体内具有很好的穿越BBB和靶向肿瘤的效果。
3、纳米药物在小鼠体内各个器官的分布情况
U87-Luc脑胶质肿瘤原位模型的建立是在BALB/c裸鼠(18～20g，6～8周龄)脑部移植肿瘤组织。选取Free Cas9/sgPLK1(Cas9-Alexa)，NP@Cas9/sgPLK1，Ang-NP@Cas9/sgPLK1三组对照，每只小鼠注射Cas9的量为0.8mg/kg，每组3只平行，对小鼠使用尾部静脉注射的方式给药，在4h的时候将小鼠处死，取出心、肝、脾、肺、肾、脑及肿瘤使用小动物成像仪荧光照射(结果如图4中(c)所示)，并将组织研碎，测量组织中的纳米粒子浓度(结果如图4中(d)所示)。
根据荧光强度可以观察纳米粒子在心、肝、脾、肺、肾、脑及肿瘤分布，Ang-NP@Cas9/sgPLK1在肿瘤部位具有较强的荧光，说明Ang-NP@Cas9/sgPLK1具有很好的肿瘤靶向效果，将组织研碎，测量组织中的纳米粒子浓度，可以精确分析Ang-NP@Cas9/sgPLK1在心、肝、脾、肺、肾、脑及肿瘤具体分布的浓度，Ang-NP@Cas9/sgPLK1在肿瘤部位的积累可以达到9.8ID％/g，相较于其他对照组有较好的积累量。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
此外，本领域的技术人员能够理解，尽管在此的一些实施例包括其它实施例中所包括的某些特征而不是其它特征，但是不同实施例的特征的组合意味着处于本发明的范围之内并且形成不同的实施例。例如，在上面的权利要求书中，所要求保护的实施例的任意之一都可以以任意的组合方式来使用。公开于该背景技术部分的信息仅仅旨在加深对本发明的总体背景技术的理解，而不应当被视为承认或以任何形式暗示该信息构成已为本领域技术人员所公知的现有技术。
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<110> 河南大学
<120> 药物载体、基于CRISPR基因编辑技术的脑靶向纳米药物及其制备方法和应用
<130>
<160> 1
<170> PatentIn version 3.3
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<211> 20
<212> DNA
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tacctacggc aaattgtgct 20

Claims (15)

1.一种药物载体，包括聚合物mPEG-P(GPMA,FPMA)和聚合物Ang-PEG-PGPMA；
所述mPEG-P(GPMA,FPMA)的结构式为：
Figure FDA0003044713760000011
所述聚合物Ang-PEG-PGPMA的结构式为
Figure FDA0003044713760000012
其中，n为35～45，x1为15～20，y为2～4，m为75～85，x2＝x1。
2.一种基于CRISPR基因编辑技术的脑靶向纳米药物，其特征在于，包括纳米粒子，所述纳米粒子由治疗药物与权利要求1所述的药物载体制备得到；所述治疗药物为核糖蛋白复合体；所述药物载体通过静电作用、 形成盐桥及氢键作用与所述核糖蛋白复合体结合。
3.根据权利要求2所述的脑靶向纳米药物，其特征在于，所述治疗药物为Cas9/sgRNA，所述Cas9/sgRNA由Cas9蛋白与sgRNA复合得到。
4.根据权利要求3所述的脑靶向纳米药物，其特征在于，所述sgRNA为靶向致癌基因PLK1的sgRNA。
5.根据权利要求4所述的脑靶向纳米药物，其特征在于，所述靶向致癌基因PLK1的sgRNA的序列如SEQ ID NO：1所示。
6.根据权利要求3所述的基于CRISPR基因编辑技术的脑靶向纳米药物，其特征在于，以摩尔份数计，所述Cas9/sgRNA中，Cas9蛋白与sgRNA的比例为1:(1～1.5)。
7.根据权利要求2-6任一项所述的基于CRISPR基因编辑技术的脑靶向纳米药物，其特征在于，以摩尔份数计，所述纳米粒子中含有：1份Cas9/sgRNA、1～3份mPEG-P(GPMA,FPMA)和4～12份Ang-PEG-PGPMA。
8.权利要求2～7任一项所述的基于CRISPR基因编辑技术的脑靶向纳米药物的制备方法，包括：
步骤a.将聚合物mPEG-P(GPMA,FPMA)、聚合物Ang-PEG-PGPMA溶解于缓冲溶液得到混合溶液A；
步骤b.在混合溶液A中加入Cas9/sgRNA，得到纳米粒子Ang-NP@Cas9/sgRNA。
9.根据权利要求8所述的制备方法，其特征在于，所述缓冲溶液选自HEPES缓冲溶液、PBS缓冲液中的任意一种。
10.根据权利要求9所述的制备方法，其特征在于，所述缓冲溶液的摩尔浓度为10～50mM。
11.根据权利要求8所述的制备方法，其特征在于，所述缓冲溶液的pH为7.4～8.0。
12.根据权利要求8所述的制备方法，其特征在于：步骤b中反应的条件为混匀室温静置。
13.根据权利要求12所述的制备方法，其特征在于，静置时间为30～120min。
14.权利要求2～7任一项所述的基于CRISPR基因编辑技术的脑靶向纳米药物在制备肿瘤治疗药物中的应用。
15.根据权利要求14所述的应用，其特征在于，所述肿瘤为脑胶质瘤。
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