CN113018435A - 一种稳定靶向光热黑磷纳米片制剂及其制备方法和应用 - Google Patents
一种稳定靶向光热黑磷纳米片制剂及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及一种稳定靶向光热黑磷纳米片制剂及其制备方法和应用,属于生物医用纳米材料技术领域。该制备方法包括以下步骤:黑磷纳米片的制备:将黑磷粉末分散至N‑甲基吡咯烷酮中,超声处理,得分散液,离心处理,得到黑磷纳米片;靶向制剂的制备:将上述黑磷纳米片分散至二甲基甲酰胺中,加入N3‑PEG‑FA和/或N3‑PEG,混合均匀,避光加热反应,得BP‑PEG和/或BP‑PEG‑FA,即为稳定靶向光热黑磷纳米片制剂。采用该方法得到的黑磷纳米片制剂具有稳定性高的特点,能在近红外激光照射下有效地杀死癌细胞,可用于肿瘤的靶向光热治疗,且能延长血液循环时间,生物相容性好,无临床应用风险,适宜广泛应用。
Description
技术领域
本发明涉及生物医用纳米材料技术领域,特别是涉及一种稳定靶向光热黑磷纳米片制剂及其制备方法和应用。
背景技术
近几十年来,尽管癌症的防治取得了令人瞩目的成就,但癌症仍然是目前最严重的疾病之一,对人类的健康造成威胁。作为传统癌症治疗方法的一种有前途的替代或补充,光热疗法(PTT)以治疗效率高、低侵袭性、毒性低等独特优势而成为医学和材料科学的研究前沿和热点。
黑磷纳米片是一种良好的光吸收剂,因其能有效地将光转化为热而被用于光热疗法,具有比石墨烯纳米片等其他二维纳米材料更高的光热转换效率和更大的消光系数。并且,高生物相容性和生物降解性使黑磷纳米片更适用于生物医学应用。
虽然黑磷(BP)作为一种新型的医用生物材料具有很大的潜力,但是黑磷在剥落成纳米薄片时,由于皱褶层的每个磷原子中都存在一对孤对电子,容易与氧气和水反应,导致黑磷降解,从而导致光学性能的损失。因此,稳定孤对电子可能是抑制黑磷降解的一种潜在策略。
最近,研究报道了几种稳定黑磷孤对电子的方法,包括表面共价功能化、非共价功能化和与过渡金属离子配位。然而这些方法仍然存在一些局限性,鉴于生物医学在人体内的应用,有效稳定的黑磷策略仍然是迫切需要的。此外,血液循环时间短易被免疫系统清除,且在癌症部位的靶向效果有待提高。
发明内容
基于此,有必要针对上述问题,提供一种稳定靶向光热黑磷纳米片制剂的制备方法,采用该方法制得的黑磷纳米片制剂具有较高的稳定性。
一种稳定靶向光热黑磷纳米片制剂的制备方法,包括以下步骤:
黑磷纳米片的制备:将黑磷粉末分散至N-甲基吡咯烷酮中,超声处理,得分散液,离心处理分散液,得到黑磷纳米片;
靶向制剂的制备:将上述黑磷纳米片分散至二甲基甲酰胺中,加入N3-PEG-FA和/或N3-PEG,混合均匀,避光加热反应,得BP-PEG和/或BP-PEG-FA,即为稳定靶向光热黑磷纳米片制剂。
研究表明,在石墨烯等无机纳米材料表面修饰PEG,可以提高材料在生理条件下的分散性,且亲水性的聚乙二醇PEG链段可以避免纳米制剂被体内的单核巨噬细胞和网状内皮系统等识别清除,从而可延长血液循环时间。此外,由于叠氮基团可与黑磷的磷原子形成氮磷共价键,磷原子为五配位饱和态,孤对电子完全成键。而稳定孤对电子可抑制黑磷降解,因此叠氮基团可以显著增强黑磷的稳定性。
在上述研究基础上,本发明人以含有叠氮基团的PEG共价修饰到黑磷表面,能稳定黑磷,增加其稳定性,从而提高了靶向光热黑磷纳米片制剂的稳定性。
并由于肿瘤细胞细胞膜表面存在过表达的叶酸受体,且叶酸是具有特定肿瘤靶向特性的B族维生素,在体内无毒副作用,具有良好的生物相容性和生物降解性等优点。通过对PEG进行叶酸修饰,可使体系特异性地靶向癌细胞,并增加肿瘤细胞对纳米制剂地摄取,进而增强纳米体系的光热治疗癌症的效果。
在其中一个实施例中,所述黑磷纳米片的制备步骤中,所述超声处理在0-4℃下进行。在上述条件下制备黑磷纳米片,具有可以避免系统的相对高温的优势
在其中一个实施例中,所述黑磷纳米片的制备步骤中,所述超声处理按照以下方法进行:以开/关周期为3-7s/3-7s为循环,超声6-8小时。以上述循环超声方式处理,有利于使大块黑磷变小变薄得到更多的黑磷纳米片。
在其中一个实施例中,所述黑磷纳米片的制备步骤中,所述离心处理按照以下方法进行:首先,将分散液以3000-5000转/分离心10-20分钟,收集上清液;之后在6000-8000转/分下离心15-25分钟,收集上清液;再在10000-14000转/分下离心15-25分钟,收集沉淀,即得黑磷纳米片。可以理解的,本领域技术人员也可以根据欲获得的黑磷纳米片尺寸大小等物理性能,对离心条件进行调整,但采取上述条件,具有较高的收率和均一性。
在其中一个实施例中,所述黑磷纳米片的最大径向尺寸为50-200nm,最大厚度为1-20nm;
所述靶向制剂的制备步骤中,所述N3-PEG和/或N3-PEG-FA中PEG的分子量为2000-10000。
在其中一个实施例中,所述靶向制剂的制备步骤中,所述反应条件为:先将反应物在液氮中冷冻、抽真空、充氩气、水冲至室温,重复2-3次,然后在130-150℃下避光加热并搅拌24-48小时。
在其中一个实施例中,所述靶向制剂的制备步骤中,所述黑磷纳米片与N3-PEG-FA和/或N3-PEG按质量比为1:40~50投料。
在其中一个实施例中,所述靶向制剂的制备步骤中,反应得到包括BP-PEG和/或BP-PEG-FA的悬浮液后,将悬浮液12000-20000转/分离心20-30分钟以获得沉淀,并用水洗沉淀2-3次,即得。
本发明还公开了一种稳定靶向光热黑磷纳米片制剂,通过上述的制备方法制备得到。
本发明还公开了上述的稳定靶向光热黑磷纳米片制剂在制备用于光热抗肿瘤纳米载体中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明的稳定靶向光热黑磷纳米片制剂的制备方法,具有简单高效的特点,且得到的黑磷纳米片制剂,包括黑磷纳米片以及官能团的聚乙二醇(PEG),聚乙二醇中的叠氮基团通过与黑磷的磷原子反应形成氮磷共价键可大大提高黑磷纳米体系的稳定性。
并且,还可通过在PEG上连接叶酸,从而利用叶酸可增强对肿瘤的靶向性。该黑磷纳米片制剂能在近红外激光照射下有效地杀死癌细胞,可用于肿瘤的靶向光热治疗,且能延长血液循环时间,生物相容性好,无临床应用风险,适宜广泛应用。
附图说明
图1为实施例1中BP和N3-PEG-FA形成复合物BP-PEG-FA的示意图;
图2为实施例1中BP-PEG-FA纳米片的透射电镜(TEM)图;
图3为实施例1中BP、BP-PEG和BP-PEG-FA纳米片的粒径分布图;
图4为实施例1中BP、BP-PEG及BP-PEG-FA的Zeta电位图;
图5为实施例1中BP和BP-PEG-FA的FT-IR图;
图6为实施例1中BP-PEG-FA的P2p和N1s的X射线光电子能谱(XPS)图;
图7为实施例2中BP、BP-PEG及BP-PEG-FA的随时间变化的体外近红外光热转化升温图;
图8为实施例2中异硫氰酸荧光素(FITC)染色的BP-PEG和BP-PEG-FA的细胞摄取图;
图9为实施例2中不同浓度BP、BP-PEG及BP-PEG-FA对HeLa肿瘤细胞体外光热治疗的定量图;其中,A为HeLa细胞未激光照射组,B为HeLa细胞近红外光(1W/cm2)照射10分钟组。
具体实施方式
为了便于理解本发明,下面将参照相关附图对本发明进行更全面的描述。附图中给出了本发明的较佳实施例。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。本文所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
以下实施例所用原料和试剂,如非特别说明,均为市售可得;以下实施例所述实验方法,如无特殊说明,均为常规方法。
实施例1
一种稳定靶向光热黑磷纳米片制剂(BP-PEG和BP-PEG-FA),通过以下方法制备得到:
(1)黑磷纳米片的制备:
在研钵中将大块黑磷片进行研磨,得到黑磷粉末,然后将30mg黑磷粉末分散在60mL N-甲基吡咯烷酮(NMP)中,利用NMP来减少氧化。
然后将混合物溶液在冰浴中用声波尖端超声处理6-8小时,具体为:以开/关周期:5s/5s为循环进行超声处理,并以冰水用来避免超声系统产生的相对高温。
随后,以4000转/分离心15分钟,以除去未剥落的块状BP,并收集上清液。之后在7000转/分下离心20分钟,除去BP沉淀并收集上清液。在12000转/分下离心上清液20分钟,收集沉淀,得到BP纳米片。
最后得到的黑磷纳米片尺寸为110nm,厚度为12nm。
(2)靶向制剂(BP-PEG和BP-PEG-FA)的制备:
分别将上述得到的BP纳米片分散在二甲基甲酰胺(DMF)中,并分别加入N3-PEG和N3-PEG-FA(来源:上海久艾生物科技有限公司),混合装于有反向冷凝器的圆底双颈烧瓶中。通过在液氮中冷冻、抽真空、充氩气、水冲至室温,重复两次,然后在140℃下避光加热48小时并搅拌反应,使黑磷纳米片带上PEG片段或PEG-FA片段,其中与N3-PEG-FA的反应过程如图1所示。
冷却后,将悬浮液12000转/分离心20分钟以获得残留物,收集沉淀并洗涤,分别得到稳定靶向光热黑磷纳米片制剂(BP-PEG和BP-PEG-FA)。
所得BP-PEG和BP-PEG-FA的特性如图2-6所示,图2为BP-PEG-FA纳米片的透射电镜(TEM)图,图3为BP、BP-PEG和BP-PEG-FA纳米片的粒径分布图,图4为BP、BP-PEG及BP-PEG-FA的Zeta电位图。从上述结果中可以看出,本实施例制备得到的BP-PEG和BP-PEG-FA纳米片的形貌和尺寸特点。
图5为BP、N3-PEG-FA和BP-PEG-FA的FT-IR图,结果显示BP和N3-PEG-FA复合后出现了1328cm-1的吸收峰,该峰为氮磷双键的特征吸收峰,说明BP和N3-PEG-FA成功地通过氮磷共价键结合。
图6为BP-PEG-FA的X射线光电子能谱,其中,A图为P2p谱,B图为N1s谱,通过拟合,结果均显示有氮磷共价健的生成,这表明N3-PEG-FA和BP发生了反应并形成了氮磷共价键。
实施例2
本实施例验证实施例1制备得到的稳定靶向光热黑磷纳米片制剂(BP-PEG及BP-PEG-FA)的稳定性。
1、方法。
将3组黑磷制剂(BP,BP-PEG,BP-PEG-FA)预先分散在水中(20.0μg/mL)。在不同的时间点(0h,12h,24h,48h,72h),测定BP,BP-PEG,BP-PEG-FA三组制剂随时间变化的体外近红外光热转化升温变化情况。
2、结果。
结果如图7所示,图7中A、B、C分别为BP,BP-PEG,BP-PEG-FA随时间变化的体外近红外光热转化升温效果。
结果显示,BP的光热转化升温能力随时间迅速降低,在3天后只能升温到32.4℃。而BP-PEG和BP-PEG-FA的升温能力稳定很多,即使在3天后其依然具有显著的升温能力,分别能升温至47.4℃、49.3℃。该结果验证了共价修饰N3-PEG-FA/N3-PEG能显著增强BP的稳定性。
实施例3
本实施例验证FITC染色的BP-PEG-FA体外细胞靶向功能和光热疗法效果。
一、体外细胞靶向功能验证。
1、方法。
以人宫颈癌细胞HeLa(来源:American Type Culture Collection ATCC,Rockville,MD,USA)来研究BP-PEG-FA的靶向功能和光热疗法效果。将HeLa细胞(5×105细胞/孔)置于共聚焦皿中培养过夜,然后加入FITC染料,BP-PEG-FA(40.0μg/mL)培养过夜,PBS洗涤三次,用多聚甲醛固定液固定细胞25分钟,PBS洗涤三次,用激光共聚焦显微镜观察荧光情况。
2、结果。
结果如图8所示,图8为异硫氰酸荧光素(FITC)染色的BP-PEG和BP-PEG-FA的细胞摄取图,从图中可以看出,BP-PEG-FA的绿色荧光比BP-PEG的绿色荧光强,说明靶向基团FA有促进纳米制剂靶向肿瘤细胞的作用。
二、体外细胞光热疗法效果验证。
1、方法。
3组黑磷制剂(BP,BP-PEG,BP-PEG-FA)对HeLa细胞的毒性通过CCK-8试验来定量。具体为:将HeLa细胞(5×104细胞/孔)接种到96孔板中并培养过夜。照射组是将不同浓度的BP,BP-PEG,BP-PEG-FA(浓度分别为0,5,10,20,40μg/mL)加入HeLa细胞中孵育4小时,之后置于近红外光下(808nm laser at 1.0W/cm2)照射10分钟。近红外光照射之后,将细胞再培养20小时,然后利用CCK-8试验定量细胞存活度。不照射组是将HeLa细胞(5×104细胞/孔)接种到96孔板中并培养过夜,细胞贴壁后加入不同浓度的BP,BP-PEG,BP-PEG-FA(浓度分别为0,5,10,20,40μg/mL),继续培养24小时,最后通过CCK-8试验检测细胞存活率。
2、结果。
结果如图9所示,图9为不同浓度BP、BP-PEG及BP-PEG-FA对HeLa肿瘤细胞体外光热治疗的定量图。其中,A为HeLa细胞未激光照射组,B为HeLa细胞近红外光(1W/cm2)照射10分钟组。从图中可以看出,不照射近红外光时,三种材料不同浓度处理的细胞的存活率(cellbiability)均高于80%,说明材料本身毒性低。
经过近红外光照射之后,三种材料处理后细胞存活率随着材料浓度的增加均降低。当BP-PEG-FA的浓度为40.0μg/mL时,将近98%的细胞死亡,但是,同样浓度的BP、BP-PEG处理的细胞存活率仍有97%和35%。说明经过叠氮和叶酸修饰之后的黑磷杀伤肿瘤细胞能力更强,光热治疗效果更好。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
1.一种稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,包括以下步骤:
黑磷纳米片的制备:将黑磷粉末分散至N-甲基吡咯烷酮中,超声处理,得分散液,离心处理分散液,得到黑磷纳米片;
靶向制剂的制备:将上述黑磷纳米片分散至二甲基甲酰胺中,加入N3-PEG-FA和/或N3-PEG,混合均匀,避光加热反应,得BP-PEG和/或BP-PEG-FA,即为稳定靶向光热黑磷纳米片制剂。
2.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述黑磷纳米片的制备步骤中,所述超声处理在0-4℃下进行。
3.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述黑磷纳米片的制备步骤中,所述超声处理按照以下方法进行:以开/关周期为3-7s/3-7s为循环,超声6-8小时。
4.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述黑磷纳米片的制备步骤中,所述离心处理按照以下方法进行:首先,将分散液以3000-5000转/分离心10-20分钟,收集上清液;之后在6000-8000转/分下离心15-25分钟,收集上清液;再在10000-14000转/分下离心15-25分钟,收集沉淀,即得黑磷纳米片。
5.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述黑磷纳米片的最大径向尺寸为50-200nm,最大厚度为1-20nm;
所述靶向制剂的制备步骤中,所述N3-PEG和/或N3-PEG-FA中PEG的分子量为2000-10000。
6.根据权利要求1-5任一项所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述靶向制剂的制备步骤中,所述反应条件为:先将反应物在液氮中冷冻、抽真空、充氩气、水冲至室温,重复2-3次,然后在130-150℃下避光加热并搅拌24-48小时。
7.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述靶向制剂的制备步骤中,所述黑磷纳米片与N3-PEG-FA和/或N3-PEG按质量比为1:40~50投料。
8.根据权利要求1所述的稳定靶向光热黑磷纳米片制剂的制备方法,其特征在于,所述靶向制剂的制备步骤中,反应得到包括BP-PEG和/或BP-PEG-FA的悬浮液后,将悬浮液12000-20000转/分离心20-30分钟以获得沉淀,并用水洗沉淀2-3次,即得。
9.一种稳定靶向光热黑磷纳米片制剂,其特征在于,通过权利要求1-8任一项所述的制备方法制备得到。
10.权利要求9所述的稳定靶向光热黑磷纳米片制剂在制备用于光热抗肿瘤纳米载体中的应用。
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