CN115350283B - 一种碳水化合物功能化纳米颗粒及其制备方法与应用 - Google Patents
一种碳水化合物功能化纳米颗粒及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及一种碳水化合物功能化纳米颗粒及其制备方法与应用,所述的纳米颗粒包括内核、壳层和表面的碳水化合物,化学式如式I所示。本发明所述的碳水化合物功能化纳米颗粒在人体内与巨噬细胞作用,高效诱导巨噬细胞M1极化,可以有效的抑制恶性肿瘤的生长,通过尾静脉注射所述的碳水化合物功能化纳米颗粒后,肿瘤相关巨噬细胞发生M1极化,肿瘤的生长被显著抑制,此外,本发明的纳米颗粒的制备方法简单,生产成本低等。
Description
技术领域
本发明属于医药技术领域,具体涉及一种碳水化合物功能化纳米颗粒及其制备方法与应用。
背景技术
巨噬细胞具有高度可塑性,响应于不同刺激信号,会获得不同的功能表型,这个过程被称为巨噬细胞的极化。根据活化表型,可将巨噬细胞分为经典活化的巨噬细胞(M1)和替代性活化的巨噬细胞(M2)。M1巨噬细胞通常由TLR配体和IFN-γ诱导激活,它们可以分泌大量促炎细胞因子、产生高活性氮和氧中间体、促进Th1反应,因而具有较高的抗微生物和抗肿瘤活性。相反,M2巨噬细胞(由IL-4/IL-13诱导激活)高表达清道夫、甘露糖等细胞表面受体以及IL-10、TGF-β等抗炎因子,它们具有免疫调节功能,可以促进组织修复和肿瘤生长。
巨噬细胞的表型转换与肿瘤的发展过程密切相关。作为免疫系统的重要组成部分,巨噬细胞本应发挥抗肿瘤功能,但肿瘤微环境信号诱导其极化为M2抗炎表型,从而促进肿瘤的生长和转移。诱导巨噬细胞复极化为M1促炎表型可以介导对肿瘤的免疫治疗。
巨噬细胞具有高吞噬活性,会主动识别并摄取进入机体的纳米颗粒,这使得纳米颗粒在调控巨噬细胞功能表型方面展现出了不可比拟的优势。但纳米颗粒本身的促炎效果通常较低,因此,对其进行功能化修饰以增强其促炎功能非常有必要。巨噬细胞的活化高度依赖于碳水化合物(如细菌或其他细胞表面的糖腭)与蛋白质(如巨噬细胞膜受体)的相互作用,因此,在纳米颗粒表面修饰碳水化合物有望增强其对M1巨噬细胞的诱导效应,从而有效抑制肿瘤的生长。
鉴于以上原因,特提出本发明。
发明内容
为了解决现有技术存在的以上问题,本发明提供了一种碳水化合物功能化纳米颗粒及其制备方法与应用,本发明所述的纳米颗粒可以高效诱导巨噬细胞M1极化,可有效的抑制恶性肿瘤的生长。
本发明的第一目的,提供了一种碳水化合物功能化纳米颗粒,所述的纳米颗粒包括内核、壳层和表面的碳水化合物,化学式如式I所示:
进一步的,所述内核为无机纳米颗粒,所述壳层为亲水性聚合物,所述表面的碳水化合物为单糖、二糖或多糖及其衍生物。
进一步的,所述的无机纳米颗粒为氧化铁、氧化锰、氧化硅、氧化铝、氧化石墨烯、金或银,所述的亲水性聚合物为聚丙烯酸、透明质酸、海藻酸、羧甲基葡聚糖或羧甲基壳聚糖,所述的单糖为葡萄糖、半乳糖或甘露糖,所述的二糖为蔗糖、乳糖或麦芽糖。
进一步的,其特征在于,所述的碳水化合物功能化纳米颗粒的粒径为5-200nm。
本发明的第二目的,提供了一种所述的纳米颗粒的制备方法,包括如下步骤:将无机纳米颗粒、氨基化碳水化合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和去离子水混合,室温下反应,透析,得到所述的纳米颗粒。
进一步的,无机纳米颗粒、氨基化碳水化合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和去离子水质量比为1:1~100:1~100:1~100:10~1000。
进一步的,室温下反应1~3天,透析介质为去离子水,透析时间为2~3天,透析袋分子量为3kDa-50kDa。
本发明的第三目的,提供了一种所述的纳米颗粒在制备治疗恶性肿瘤药物中的应用。
进一步的,所述的恶性肿瘤包括原发性或继发性肝癌、乳腺癌、黑色素瘤、肺癌、胃癌中的一种或几种。
与现有技术相比,本发明的有益效果为:
(1)由于巨噬细胞的活化高度依赖于其表面蛋白质(如膜受体)与病原体表面碳水化合物的相互作用,本发明将碳水化合物修饰在纳米颗粒表面后可使得该纳米颗粒具有类似于细菌等病原体的结构,从而能高效激活巨噬细胞的M1极化;
(2)本发明所述的碳水化合物功能化纳米颗粒在人体内与巨噬细胞作用,高效诱导巨噬细胞M1极化,可以有效的抑制恶性肿瘤的生长,通过尾静脉注射所述的碳水化合物功能化纳米颗粒后,肿瘤相关巨噬细胞发生M1极化,肿瘤的生长被显著抑制,此外,本发明的纳米颗粒的制备方法简单,生产成本低等。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的一些实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1是本发明的葡萄糖功能化氧化铁纳米颗粒的透射电镜图;
图2是本发明的葡萄糖功能化氧化铁纳米颗粒的扫描电镜图;
图3是本发明的葡萄糖功能化氧化铁纳米颗粒的红外图;
图4是本发明的葡萄糖功能化氧化铁纳米颗粒的能谱图;
图5是巨噬细胞M1极化相关基因表达图;
图6是小鼠皮下B16黑色素瘤生长抑制曲线;
图7是小鼠皮下LLC肺癌肿瘤生长抑制曲线。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
实施例1
葡萄糖功能化氧化铁纳米颗粒的制备
本实施例的纳米颗粒的内核为四氧化三铁,壳层为聚丙烯酸,表面为葡萄糖,具体制备方法如下:取14ml氧化铁纳米颗粒水溶液加入到50ml圆底反应瓶中,其中氧化铁纳米颗粒质量为32mg,然后在快速搅拌下向其中依次加入3mL N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC,29mg/mL)和3mL N-羟基琥珀酰亚胺(NHS,35mg/mL)水溶液。同时,称取194mg D-氨基葡萄糖盐酸盐,将其溶解到4mL 10mg/mL的氢氧化钠水溶液中以脱去其盐酸盐成分,得到D-氨基葡萄糖。EDC/NHS活化30分钟后,将D-氨基葡萄糖溶液加入到上述氧化铁纳米颗粒溶液中,室温下搅拌反应24小时。停止反应后用超纯水透析3天(Mw截止值为3.5kDa),去除未反应成分。通过旋转蒸发仪除去大量水后,得到浓缩液,保存至4℃备用,得到葡萄糖功能化氧化铁纳米颗粒分散液。
实施例2
葡萄糖功能化银纳米颗粒的制备
本实施例的纳米颗粒的内核为银,壳层为聚丙烯酸,表面为葡萄糖,具体制备方法如下:取10mL银纳米颗粒水溶液加入到50ml圆底反应瓶中,其中银纳米颗粒质量为28mg,然后在快速搅拌下向其中依次加入3mL N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC,13mg/mL)和3mL N-羟基琥珀酰亚胺(NHS,15mg/mL)。同时,称取86mg D-氨基葡萄糖盐酸盐溶解到2mL10mg/mL的氢氧化钠水溶液中脱盐酸盐,得到D-氨基葡萄糖。EDC/NHS活化30分钟后,将D-氨基葡萄糖溶液加入到上述银纳米颗粒溶液中,室温下搅拌反应24小时。停止反应后用超纯水透析3天(Mw截止值3.5kDa),去除未反应成分。通过旋转蒸发仪除去大量水后,得到浓缩液,保存至4℃备用,得到葡萄糖功能化银纳米颗粒分散液。
实施例3
葡萄糖功能化金纳米颗粒的制备
本实施例的纳米颗粒的内核为金,壳层为聚丙烯酸,表面为葡萄糖,具体制备方法如下:取10mL金纳米颗粒水溶液加入到50ml圆底反应瓶中,其中金纳米颗粒质量为50mg,然后在快速搅拌下向其中依次加入3mL N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC,44mg/mL)和3mL N-羟基琥珀酰亚胺(NHS,27mg/mL)。同时,称取150mg D-氨基葡萄糖盐酸盐溶解到4mL 10mg/mL的氢氧化钠水溶液中脱盐酸盐,得到D-氨基葡萄糖。EDC/NHS活化30分钟后,将D-氨基葡萄糖溶
液加入到上述金纳米颗粒溶液中,室温下搅拌反应24小时。停止反应后用超纯水透析3天(Mw截止值3.5kDa),去除未反应成分。通过旋转蒸发仪除去大量水后,得到浓缩液,保存至4℃备用,得到葡萄糖功能化金纳米颗粒分散液。
实施例4
甘露糖功能化氧化铁纳米颗粒的制备
本实施例的纳米颗粒的内核为四氧化三铁,壳层为透明质酸,表面为甘露糖,具体制备方法如下:取10ml氧化铁纳米颗粒水溶液加入到50ml圆底反应瓶中,其中氧化铁纳米颗粒质量为25mg,然后在快速搅拌下向其中依次加入3mL N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC,23mg/mL)和3mL N-羟基琥珀酰亚胺(NHS,27mg/mL)水溶液。同时,称取152mg D-氨基甘露糖盐酸盐,将其溶解到3mL 10mg/mL的氢氧化钠水溶液中以脱去其盐酸盐成分,得到D-氨基甘露糖。EDC/NHS活化30分钟后,将D-氨基甘露糖溶液加入到上述氧化铁纳米颗粒溶液中,室温下搅拌反应24小时。停止反应后用超纯水透析3天(Mw截止值为3.5kDa),去除未反应成分。通过旋转蒸发仪除去大量水后,得到浓缩液,保存至4℃备用,得到甘露糖功能化氧化铁纳米颗粒分散液。
实施例5
半乳糖功能化金纳米颗粒的制备
本实施例的纳米颗粒的内核为金,壳层为海藻酸,表面为半乳糖,具体制备方法如下:取15mL金纳米颗粒水溶液加入到50ml圆底反应瓶中,其中金纳米颗粒质量为80mg,然后在快速搅拌下向其中依次加入3mL N-(3-二甲基氨基丙基)-N'-乙基碳二亚胺盐酸盐(EDC,70mg/mL)和3mL N-羟基琥珀酰亚胺(NHS,43mg/mL)。同时,称取240mg D-氨基半乳糖盐酸盐溶解到6.5mL 10mg/mL的氢氧化钠水溶液中脱盐酸盐,得到D-氨基半乳糖。EDC/NHS活化30分钟后,将D-氨基半乳糖溶液加入到上述金纳米颗粒溶液中,室温下搅拌反应24小时。停止反应后用超纯水透析3天(Mw截止值3.5kDa),去除未反应成分。通过旋转蒸发仪除去大量水后,得到浓缩液,保存至4℃备用,得到半乳糖功能化金纳米颗粒分散液。
试验例1
本试验例将实施例1制备的葡萄糖功能化氧化铁纳米颗粒进行如下性能的检测
1、将葡萄糖功能化氧化铁纳米颗粒经透射电镜(TEM)检测:
取适量葡萄糖功能化氧化铁纳米颗粒分散液稀释至葡萄糖功能化氧化铁纳米颗粒吸取10μL滴至铜网表面,室温下自然风干,然后将样品置于透射电镜下拍摄,结果如图1所示,氧化铁纳米颗粒的晶核在5-10纳米左右,呈现类球形形貌。
2、将葡萄糖功能化氧化铁纳米颗粒进行扫描电镜(SEM)检测:
取适量葡萄糖功能化氧化铁纳米颗粒分散液稀释至1mg/mL,吸取50μL滴至制样台上的单晶硅表面,室温下自然风干,然后将样品置于扫描电镜下拍摄,结果如图2所示,氧化铁纳米颗粒的整体粒径在10-30纳米左右。
3、葡萄糖功能化氧化铁纳米颗粒的红外检测:
取葡萄糖功能化氧化铁纳米颗粒分散液冻干,取2mg左右待测样品与溴化钾混匀后放入磨具中,压制成透明薄片后进行检测。根据样品的红外吸收峰分析样品的化学键和官能团,结果如图3所示,在1650cm-1处出现了酰胺键的峰,表明葡萄糖的成功修饰。
4、葡萄糖功能化氧化铁纳米颗粒元素分析:
取葡萄糖功能化氧化铁纳米颗粒分散液冻干,然后将粉末涂覆在导电胶上,通过SEM的能谱(Energy dispersive spectrometer,EDS)和元素分析功能对其元素进行定性和定量的分析,结果如图4所示,可以看出葡萄糖修饰后,铁的含量较低。
5、葡萄糖功能化氧化铁纳米颗粒诱导巨噬细胞M1极化相关基因上调:
通过实时荧光定量PCR(qPCR)平菇巨噬细胞M1极化相关基因上调,具体步骤如下:
(1)RNA的提取和纯化
①巨噬细胞RAW264.7在培养24小时后,弃去培养基,加入1mL Trizol裂解细胞,10分钟后,将其转移到无RNA酶污染的EP管中。
②加入200μL氯仿,上下颠倒混匀后静置2~3分钟,4℃,12000rpm下离心15分钟。
③吸取上层溶液,再加入等体积的异丙醇,缓慢颠倒混匀后静置10~15分钟,然后4℃,12000rpm下离心15分钟。
④缓慢弃掉上清,加入1ml 75%的乙醇洗涤,以去除蛋白质等杂质,4℃,10000rpm下离心10分钟。
⑤弃上清,并用枪头吸去剩余液体,室温下放置5分钟左右让乙醇自然挥发,当看到白色的RNA沉淀逐渐变的透明时加入DEPC水溶解RNA。
(2)RNA逆转录
根据iScript cDNA合成试剂盒说明书将RNA逆转录为cDNA。所有的操作过程都在冰上完成,反应体系为20μL,具体如表1:
表1
5xiScriptRTSupermix | 4μL |
RNA(1μg)模板+Nuclease-free水 | 16μL |
热循环参数设置如表2所示。
表2
反应阶段 | 温度(℃) | 时间(min) |
启动(Priming) | 25 | 5 |
延伸(Reversetranscription) | 46 | 20 |
终止(RTinactivation) | 95 | 1 |
(3)qPCR
根据SsoFast EvaGreen Supermixes说明书的方法进行目标基因的qPCR反应,反应体系为10μL体系,具体如表3。
表3
SosoFastEvaGreenSupermix | 5μL |
Forwardprimer(500nM) | 0.5μL |
Reverseprimer(500nM) | 0.5μL |
DNAtemplate | 1μL |
RNase/DNase-freewater | 3μL |
qPCR引物序列见表4。
表4
基因 | ForwardPrimer(5'-3') | ReversePrimer(5'-3') |
CD86 | TTGTGTGTGTTCTGGAAACGGAG | AACTTAGAGGCTGTGTTGCTGGG |
NOS2 | CACGGACGAGACGGATAG | CACTGACACTTCGCACAAA |
TNF-α | CTGAACTTCGGGGTGATC | TCCTCCACTTGGTGGTTT |
CD206 | AGGGTGCGGTACACTAAC | CAACACGGTATGACAGAAA |
Arginase1 | AAGACAGCAGAGGAGGTG | AGTCAGTCCCTGGCTTAT |
IL-10 | ACTGCTAACCGACTCCTT | TCCACTGCCTTGCTCTTA |
IL-1β | AGCACCTTCTTTTCCTTC | TGCCGTCTTTCATTACAC |
IL-6 | GCCTTCTTGGGACTGATG | CTGGCTTTGTCTTTCTTCTT |
IL-12p40 | GGACATCATCAAACCTGACC | AGGGAGAAGTAGGAATGTGG |
SOCS3 | GCCAGTCCTAGTCATCTCT | GCTTCTCCATCACCTCCT |
β-actin | GCACCACACCTTCTACAA | TACGACCAGAGGCATACA |
结果如图5所示,相较于生理盐水和未修饰的氧化铁纳米颗粒处理组,葡糖功能化氧化铁纳米颗粒更高效地上调了M1巨噬细胞相关基因CD86、NOS2和TNF-α的表达,表明葡糖糖修饰有效增强了氧化铁纳米颗粒对M1巨噬细胞的诱导效应。
6、葡萄糖功能化氧化铁纳米颗粒诱导巨噬细胞M1极化后,抑制小鼠黑素瘤生长试验
首先在C57小鼠右侧腋窝后方注射5×105个B16黑素瘤细胞,建立小鼠黑色素瘤肿瘤模型,待肿瘤生长至一定体积,通过尾静脉注射葡萄糖功能化氧化铁纳米颗粒,注射剂量为5mg Fe/kg,间隔三天注射一次。在给药期间,每天监测肿瘤体积的变化。结果如图6所示,葡糖功能化氧化铁纳米颗粒显著抑制了肿瘤的生长,抑制效果显著优于未修饰的氧化铁纳米颗粒。表明葡糖糖修饰有效增强了氧化铁纳米颗粒对肿瘤的抑制功能。。
7、葡萄糖功能化氧化铁纳米颗粒诱导巨噬细胞M1极化后,抑制小鼠肺癌肿瘤生长试验
首先在C57小鼠右侧腋窝后方注射100μL LLC肺癌细胞(1×106个)和葡萄糖功能化氧化铁纳米颗粒(100μg Fe)的共混液,然后每天监测肿瘤体积的变化,结果如图7所示,葡糖功能化氧化铁纳米颗粒高效抑制了肿瘤的生长,抑制效果同样显著优于未修饰的氧化铁纳米颗粒,再次证明葡糖糖修饰能有效增强氧化铁纳米颗粒对肿瘤的抑制功能。
本发明人也对其他实施例做了上述试验,结果基本一致,由于篇幅有限,不再一一列举。
以上所述,仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到变化或替换,都应涵盖在本发明的保护范围之内。因此,本发明的保护范围应以所述权利要求的保护范围为准。
Claims (6)
1.一种碳水化合物功能化纳米颗粒,其特征在于,所述的纳米颗粒包括内核、壳层和表面的碳水化合物,化学式如式I所示:
式I;
所述内核为无机纳米颗粒,所述壳层为亲水性聚合物,所述表面的碳水化合物为葡萄糖;
所述无机纳米颗粒为四氧化三铁;
所述亲水性聚合物为聚丙烯酸。
2.根据权利要求1所述的碳水化合物功能化纳米颗粒,其特征在于,所述的碳水化合物功能化纳米颗粒的粒径为5~200nm。
3.一种权利要求1-2任意一项所述的纳米颗粒的制备方法,其特征在于,包括如下步骤:将内核为无机纳米颗粒和壳层为亲水性聚合物的颗粒、氨基化碳水化合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和去离子水混合,室温下反应,透析,得到所述的纳米颗粒。
4.根据权利要求3所述的纳米颗粒的制备方法,其特征在于,内核为无机纳米颗粒和壳层为亲水性聚合物的颗粒、氨基化碳水化合物、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和去离子水质量比为1:1~100:1~100:1~100:10~1000。
5.根据权利要求3所述的纳米颗粒的制备方法,其特征在于,室温下反应1~3天,透析介质为去离子水,透析时间为2~3天,透析袋分子量为3kDa-50kDa。
6.一种权利要求1-2任一所述的纳米颗粒或权利要求3-5任一方法制备的纳米颗粒在制备治疗恶性肿瘤药物中的应用;
所述的恶性肿瘤为原发性或继发性肝癌、乳腺癌、黑色素瘤、肺癌、胃癌中的一种或几种。
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