CN113082223A - 纳米载体及含其的药物递送系统 - Google Patents
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Abstract
本发明提供了载药用纳米载体，所述纳米载体的靶向配体包括特异性靶向M2型巨噬细胞的多肽。本发明还提供了含其的药物递送系统。本发明的载体成功实现了特异性靶向M2型巨噬细胞，载体的毒性/免疫原性低,稳定性、缓释好，载重极化M2的药后，能大幅提高M2重极化率，提高抗肿瘤效果。
Description
技术领域
本发明涉及一种纳米载体及其应用。
背景技术
纳米载体是一种具有纳米尺度的亚微粒药物载体输送系统，可以通过包载等方式将药物分子包封在保护性壳状结构内。经检索，尽管目前利用纳米载体将药物递送至M2型巨噬细胞的研究已有很多，但是这些药物载体的递送并不是特异性递送因此存在药效差的问题,抑或是此类纳米药物存在引起全身性免疫反应等毒副作用的风险。
发明内容
本发明克服了现有技术的不足，一方面提供了一种药物纳米载体，所述纳米载体的靶向配体包括特异性靶向M2型巨噬细胞的多肽，多肽的序列可为YEQDPWGVKWWY。
可选地，所述纳米载体载药用的递送材料为聚乳酸-羟基乙酸共聚物(PLGA)。
可选地，所述纳米载体包含有用于助其减少被给药对象的网状内皮系统RES捕获的囊泡，载药用的递送材料被内置在该囊泡中；当然，使用载体时，药物亦随递送材料内含在所述囊泡中。
囊泡在本专利中为本领域常见涵义，意指类似于细胞膜那样，由两亲性分子形成的具有封闭双层结构的分子有序组合体。
任选地，所述囊泡的膜表面含有CD47蛋白。
任选地，所述囊泡源于高表达CD47蛋白细胞的细胞膜，高表达意指该细胞比给药对象正常细胞，在细胞膜表面含有更高水平的CD47，多为癌细胞，如黑色素瘤细胞的细胞膜，可选为B16-OVA细胞的细胞膜。具体制备时，可直接使用细胞匀浆裂解得到的细胞膜，将其挤成泡状。
任选地，所述多肽通过与其共价连接(如通过形成酰胺键而相连)的疏水链而嵌接在囊泡膜的外表面，该疏水链可为二硬脂酰基磷脂酰乙醇胺(DSPE)的碳链，如共价连接通过所述多肽与DSPE–PEG-NH2反应实现，DSPE–PEG-NH2为DSPE、聚乙二醇PEG(PEG可为PEG2000)、-NH2依序相连的结构。
任选地，所述给药对象为哺乳动物，如人。
另一方面，本发明提供了递送系统，该系统包含被递送的药物及上述纳米载体。
任选地，所述药物能够重极化M2型巨噬细胞，如为瑞喹莫德(resiquimod)R848。
可补充地，所述系统中药物与其递送材料形成的纳米颗粒NP可为水包油(O/W)结构，制备可用水包油乳化法液，乳化剂可为聚乙烯醇，所述递送材料与药物接触以包载药物前，递送材料可用二氯甲烷预溶解，药物可用DMSO预溶解，所投药物与所投递送材料的质量比可为1：10-400，如为1：10-100。递送材料与药物接触后，可通过超声混匀，离心得到NP。NP制成后，用囊泡包裹NP，将所述多肽通过与其共价连接的疏水链嵌接在囊泡膜外表，该疏水链可为二硬脂酰基磷脂酰乙醇胺DSPE碳链，共价连接可为形成酰胺键而相连，如通过所述多肽与DSPE–PEG-NH2缩合实现，缩合可使用碳二亚胺，如EDC。
本发明的有益效果为：
本发明的载体成功实现了特异性靶向M2型巨噬细胞，载体的毒性/免疫原性低,稳定性、缓释好，载重极化M2的药后，能大幅提高M2重极化率，提高抗肿瘤效果。
附图说明
图1示范本专利载体NP-R@M-T各模块的结构及其制备；
图2比较了本专利载体与其他对照组对M2型巨噬细胞表型转化的影响(细胞水平)；
图3比较了本专利载体与其他对照组处理后，M1型巨噬细胞因子分泌的水平；
图4比较了本专利载体与其他对照组对M2型巨噬细胞表型转化的影响(mRNA水平)；
图5比较了对小鼠体内各给药形式的抗肿瘤效果，其中5A为M1/M2型巨噬细胞浸润比例的流式统计，5B、5C均是组织中分泌IFN-γ的CD8+T细胞的比例的统计，分别涉及脾脏、引流淋巴结；
各图中涉及的统计学标识含义：*P<0.05，**P<0.01，***P<0.001。
具体实施方式
为节约篇幅，对本领域技术人员公知、熟悉或惯常操作的技术细节，本专利有所省略，如无特别说明，下面所用试剂、生物材料、培养基和溶液均为本领域常用、公众可以得到或市售，或者通过常规制备能够得到的物品。下面为详细示例说明本发明，而不应该为限制本发明的范围，分章节描述如下：
第1章可能涉及的英文缩写表
续表1
第2章NP-R@M-T纳米载体的制备与表征
2.1部分试剂来源
DSPE-PEG(2000)-NH2 Avanti Polar Lipids
M2pep(YEQDPWGVKWWY) 本实验室合成
ScM2pep(WEDYQWPVYKGW) 本实验室合成
2.2实验方法
2.2.1合成PLGA纳米颗粒(NP)以及该NP包载R848的复合物NP-RPLGA纳米颗粒的合成采用“水包油”的乳化方法，具体合成步骤如下：
(1)用分析天平称取10mg的PLGA粉末溶于含400μL DCM的EP管中，充分震荡至PLGA完全溶解，将PLGA溶液转移至15mL离心管里。
(2)继续加入3.6mL的2.5％的PVA水溶液。若制备包载R848的NP-R，则另加入DMSO溶解好的0.1mg R848(细胞实验用量)或1mg R848(动物实验用量)，比例平衡实验见后。
(3)将15mL离心管固定在盛有冰的200mL烧杯里，放进超声波细胞破碎仪里，以3son/3s off的工作频率超声1分钟后，补加2mL 2.5％的PVA水溶液。
(4)放入磁力搅拌转子，置于磁力搅拌器上，以500rpm/min的频率过夜搅拌至DCM完全挥发。
(5)DCM挥发完后，4000rpm/min,10min,4℃离心去掉大颗粒(沉淀)，取上清至干净的1.5mL EP管里，14000rpm/min，30min，4℃，离心，弃上清，收集纳米颗粒沉淀，并用200μL双蒸水超声重悬,补加体积至1mL后，再次离心，以洗去残留的PVA以及未包裹进PLGA里的R848等杂质。
(6)14000rpm/min，30min，4℃反复离心3次后，移入离心管中。
为平衡载药效果，本研究分别制备了PLGA与R848质量比为400:1，200:1，100:1，50:1，20:1，10:1的NP-R，分离获得各纳米载体前，检测上清中游离药物的量并计算：
包封率(％,w/w)＝(R848的总投入量-上清液中R848含量)/R848的总投入量×100
载药量(％,w/w)＝(R848的总投入量-上清液中R848含量)/(R848的包封量+载体的总投入量)×100
最终，我们选取了PLGA:R848的比值为100:1进行后续的细胞实验，动物实验比值为10：1。
2.2.2提取B16-OVA细胞膜
(1)收集细胞：挑选处于对数生长期的细胞用于细胞膜提取。
(2)细胞匀浆裂解：使细胞充分破碎。
(3)去除细胞核和未破碎的细胞
(4)沉淀细胞膜碎片。
2.2.3纳米载体NP@M以及NP-R@M的构建
(1)向细胞膜沉淀中加入PBS，超声重悬细胞膜沉淀后，用装有400nm聚碳酸酯薄膜的挤出机挤压细胞膜悬液11次，获得细胞膜囊泡。
(2)取制备好的NP和NP-R纳米载体与细胞膜囊泡进行充分混合，随后用装有200nm聚碳酸酯薄膜的挤出机挤压混合液11次，以生成用细胞膜包裹的纳米载体，即NP@M和NP-R@M纳米复合物。
2.2.4 DSPE-M2pep以及DSPE-ScM2pep的合成
(1)用配置好的MES缓冲液(浓度为0.1M，pH6.0)溶解NHS和EDC，调整浓度为2mg/mL。
(2)称取M2pep(2μM)和ScM2pep(2μM)，与1.1mg NHS(保证反应体系里NHS终浓度为2mM)和0.4mg EDC(保证反应体系里EDC终浓度为5mM)，总体系为1mL，共孵育15min。
(3)称取两份DSPE-PEG(2000)-NH2(2.5μM)分别与M2pep、Sc M2pep反应2h，终止反应，共价产物依次简称为DSPE-M2pep、DSPE-ScM2pep。
2.2.5NP-R@M-T以及NP-R@M-S纳米载体的构建
(1)取制备好的NP-R@M纳米载体2mg，平均分成两份，分别与20μg的DSPE-M2pep和20μg DSPE-ScM2pep在4℃冰箱里置于磁力搅拌器上进行搅拌。
(2)充分搅拌2小时后，即得到NP-R@M-T以及NP-R@M-S纳米复合物。以NP-R@M-T为例，上述各步制备过程总结如图1。
2.2.6 NP-R@M-T的表征
(1)纳米复合物Zeta电位和粒径检测：纳米复合物样品(1mg/mL)，利用马尔文粒度电位仪检测不同的纳米粒子的电位和粒径分布。
(2)纳米复合物TEM成像
2.3实验结果
2.3.1 NP-R的药物包封率和载药量
表2.1纳米载体NP-R在不同PLGA:R848质量比下的包封率和载药量
2.3.2 NP-R以及NP-R@M-T的体外药物释放
本实验采用透析法考察，NP-R在前12h内的释药速度较快，12h后释放速度有所下降，且120h内的累计释放率达21.9±2.57％，相比于NP-R，NP-R@M-T的体外释药速度略有减慢，但二者相比并无显著性差异。
2.3.3 DSPE-M2pep以及DSPE-ScM2pep的合成
合成得到的产物利用红外光谱仪进行鉴定后，确认成功：M2pep和DSPE-PEG(2000)-NH2合成所得到的产物有一个O-H基的特征吸收峰(3291cm-1)。
2.3.4 NP-R@M-T的表征
NP-R@M-T的平均粒径在188nm左右，Zeta电位在-9.7mV左右。另一方面，细胞膜成功地覆盖在NP-R载体上，TEM结果进一步表明成功：NP-R@M-T呈圆形或类圆形，且表现出明显的核壳结构，其均匀的脂质双分子层的厚度约为10nm。电位说明配体成功连接上纳米载体。
此外，我们还证明NP-R@M-T能够在含10％的血清的PBS溶液里于37℃下稳定存在7天。最后，我们利用SDS-PAGE分析了NP-R@M的蛋白质成分，结果表明纯的细胞膜蛋白条带以及纳米载体上的蛋白条带一致。
第3章NP-R@M-T体外毒性、摄取和体内靶向性研究
对制备的纳米载体表征完后，我们接着考察了纳米载体在体内外摄取的情况以及载体的细胞毒性，结果如下：
3.3.1纳米载体的细胞毒性评价
采用MTT比色法来测定构建的不同纳米载体对B16-OVA细胞的毒性大小，载体中的R848含量从0.4μg/mL～1.6μg/mL变化时，NP-R@M-T各载体均没有毒性作用。
3.3.2纳米载体体外摄取实验
在上章所制载药载体的基础上，进一步用亲脂性荧光染料DIO标记B16-OVA细胞膜，形成荧光纳米载体DIO-NP-R@M-T和DIO-NP-R@M-S。从共聚焦荧光成像、流式细胞术的结果上看，M2型巨噬细胞在3小时摄取DIO-NP-R@M-T的量要比DIO-NP-R@M-S更多。
3.3.3纳米载体体内摄取实验
我们构建了黑色素瘤B16-OVA小鼠皮下移植瘤模型,在上章所制载药载体的基础上，进一步将荧光染料DID包裹入NP以制备成荧光纳米载体NP-D@M-T和NP-D@M-S。实验结果显示，相比于M1型TAMs，DCs，B16-OVA，NP-D@M-T在6h时更多的被M2型巨噬细胞吞噬，且在24h时，吞噬荧光载体的M2型巨噬细胞数量有明显的增加，吞噬载体的平均细胞数从46％增加到了58％。另一方面，从平均荧光强度的流式统计结果分析，由于载体修饰了M2pep，相比于NP-D@M-S，M2型巨噬细胞不论是在6h还是在24h都摄取了更多的NP-D@M-T。
第4章纳米载体对M2型巨噬细胞表型转化的影响
4.3.1纳米载体对M2型巨噬细胞表型转化的影响(细胞水平)
为了考察不同组纳米载体将M2型巨噬细胞重极化为M1型巨噬细胞的能力，我们选择CD11c作为M1型巨噬细胞标志物进行流式细胞术分析，由图2可见，与对照组NP-R@M-S等相比，NP-R@M-T能够将更多的M2型巨噬细胞极化为M1型巨噬细胞。
4.3.2纳米载体对M2型巨噬细胞表型转化的影响(蛋白水平)
收集不同处理组的细胞培养上清，ELISA检测IL-12p70的分泌情况如图3。
4.3.3纳米载体对M2型巨噬细胞表型转化的影响(mRNA水平)
实时荧光定量PCR技术检测不同纳米载体刺激24h后细胞中TNFα、iNOS、CD86以及IL12 mRNA的表达情况,qRT-PCR实验结果如图4所示。
第5章纳米载体对治疗黑色素瘤的评价
5.3.1纳米载体对黑色素瘤生长的抑制作用
我们构建了黑色素瘤B16-OVA小鼠皮下移植瘤，分别通过尾静脉注射PBS、NP-R、NP-R@M-S、NP-R@M-T和R848，给药期间，记录小鼠的体重和肿瘤体积变化。治疗组小鼠体重与PBS组小鼠体重并无显著性差异，在接种肿瘤后17天，NP-R@M-T治疗组的肿瘤体积相比于PBS组缩小了82％，而NP-R@M-S治疗组相比于PBS组仅缩小了65％。同时，发现NP-R@M-T治疗组的小鼠存活时间最长，与PBS组相比具有显著的差异。
5.3.2纳米载体的抗肿瘤效果
流式结果表明与PBS对照组相比，NP-R@M-S减少了肿瘤部位37％的M2型巨噬细胞(CD45+CD11b+F4/80+CD206+)，然而，我们在NP-R@M-T治疗组小鼠肿瘤里观察到了更显著的M2型巨噬细胞浸润百分比减少现象(51％)。同时，我们也检测了M1/M2的在肿瘤部位浸润比值情况，结果如图5A所示，我们可以发现，修饰了M2pep的纳米载体NP-R@M-T能更高效地改善肿瘤部位M1/M2的浸润比例。
与PBS组相比，NP-R@M-T组肿瘤浸润的CD8+T细胞(CD45+CD3+CD8+)增加了2.4倍，而NP-R@M-S治疗组CD8+T细胞浸润比例明显少于NP-R@M-T组。同时，NP-R@M-T治疗组CD8+T细胞活化(CD69是CD8+T细胞活化的标志物)的比例与PBS组相比，明显增加了3.7倍。
此外，我们分离荷瘤小鼠肿瘤引流淋巴结和脾脏进行ex vivo实验分析浸润的T细胞IFN-γ的分泌情况，结果如图5B～5C所示。
5.3.3纳米载体对小鼠器官的毒副作用
为了评价纳米载体对小鼠器官的毒副作用，我们取各组治疗后小鼠的心、肝、脾、肺、肾等主要脏器进行组织病理学分析。标准H&E染色显示，治疗后小鼠主要器官组织与对照组相比并无异常。因此，我们认为给药剂量在10mg/kg时，纳米载体对小鼠主要脏器无毒副作用。
Claims (10)
1.载药用纳米载体，所述纳米载体的靶向配体包括特异性靶向M2型巨噬细胞的多肽。
2.如权利要求1所述的纳米载体，其特征是，所述多肽的序列为YEQDPWGVKWWY，所述纳米载体可包含有用于助其减少被给药对象的网状内皮系统捕获的囊泡，载药用的递送材料被内置在该囊泡中；独立可选地，纳米载体载药用的递送材料为聚乳酸-羟基乙酸共聚物PLGA。
3.如权利要求2所述的纳米载体，其特征是，所述囊泡的膜表面含有CD47蛋白。
4.如权利要求2或3所述的纳米载体，其特征是，所述囊泡源于高表达CD47蛋白细胞的细胞膜，如黑色素瘤细胞的细胞膜，优选为B16-OVA细胞的细胞膜。
5.如权利要求2所述的纳米载体，其特征是，所述多肽通过与其共价连接(如通过形成酰胺键而相连)的疏水链而嵌在囊泡膜外表，该疏水链可为二硬脂酰基磷脂酰乙醇胺DSPE碳链，如共价连接通过所述多肽与DSPE-PEG-NH2缩合实现，DSPE-PEG-NH2为DSPE、聚乙二醇PEG(PEG可为PEG2000)、-NH2依序相连的结构。
6.如权利要求2所述的纳米载体，其特征是，所述给药对象为哺乳动物，如人。
7.药物递送系统，该系统包含任一在先权利要求所述的纳米载体及被该载体递送的药物。
8.如前一权利要求所述的系统，其特征是，所述药物能够重极化M2型巨噬细胞，如为瑞西莫德。
9.如前一权利要求所述的系统，其特征是，所述系统的制备工艺包括：采用水包油乳化法将所述药物与其递送材料制成纳米颗粒NP，用囊泡包裹NP，将所述多肽通过与其共价连接的疏水链嵌在囊泡膜外表。
10.如前一权利要求所述的系统，其特征是，所述乳化法使用的乳化剂为聚乙烯醇，所述递送材料与药物接触以包载药物前，递送材料可用二氯甲烷预溶解，药物可用DMSO预溶解，所投药物与所投递送材料的质量比可为1:10-400，如为1:10-100。
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