CN115040493A - 一种核酸纳米粒的制备方法 - Google Patents
一种核酸纳米粒的制备方法 Download PDFInfo
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- CN115040493A CN115040493A CN202210602545.4A CN202210602545A CN115040493A CN 115040493 A CN115040493 A CN 115040493A CN 202210602545 A CN202210602545 A CN 202210602545A CN 115040493 A CN115040493 A CN 115040493A
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Abstract
本发明涉及材料技术领域,公开了一种核酸纳米粒的制备方法。本发明采用改良型的超声方法,在两种液体快速混合的过程中,采用超声波进行辅助混合,使两种溶液能够快速均匀地进行混合,在混合的瞬间,核酸和载体能够快速结合并生成纳米粒,纳米粒的生成时间大约为一分钟左右。生成的纳米粒均匀性好,勿需经过更进一步的均质。该方法反应时间短,纳米粒均匀性高,重复性好,可放大生产。制备的核酸纳米粒可以用作核酸治疗药物,核酸疫苗,核酸农药等。
Description
技术领域
本发明涉及材料技术领域,尤其涉及一种核酸纳米粒的制备方法。
背景技术
目前核酸纳米制剂有很多种制备方法,但这些方法通常存在着多种缺点。传统的溶剂注入法可用于制备脂质体,分为乙醇注入法和乙醚注入法,通常用来包裹油溶性药物,少数可用于水溶性药物的包裹,但通常载药率比较低。并且该方法采用磷脂作为载体化合物,将磷脂溶于有机溶剂中,该方法的形成的脂质体层中通常带有空隙,空隙中含有有机溶剂,后期很难将其完全除去。同时该方法对包载药物有限制,通常用于油溶性药物的包载,将油溶性药物和磷脂化合物共同溶解于有机溶剂中,少数改进的方法可用于水溶性药物的包载。而对于核酸化合物的包载,通常得到的纳米粒子粒径不稳定、mRNA包封率较低,因此很少有人使用。目前较为先进的技术,如生产中可以采用高压微射流,微流控等,实验室研发中的则可采用微流控法,挤出器挤出法,冷冻干燥法等,能够得到包载率高,粒径均匀且可控的核酸纳米粒。但这些方法中高压微射流和微流控的设备昂贵,研发和生产成本大;而挤出器则耗材成本昂贵,不宜放大。其它的纳米制剂,如核酸胶束,核酸/多肽纳米粒,核酸/聚合物纳米粒等的制备也存在着诸多缺点。
传统的超声分散也是一种在纳米材料领域广泛应用的一种方法,能够增加搅拌混合作用,可用于纳米粒的再分散。也可用于大颗粒的解团聚、细化,较大的纳米粒经过长时间的超声,能够得到粒径更小更均匀的纳米粒。该方法通常用于包载水溶性药物,将水溶性药物溶于水溶液,多种载体材料以及油溶性药物溶于有机溶剂中,首先采用搅拌蒸发去除有机溶剂,然后进行超声波处理,可得脂质体纳米制剂。但是该方法过程繁琐,所需时间较长,且获得的脂质体制剂的均匀性不高。并且,包括核酸在内的生物分子,通常不适用于长时间的超声。因为超声对生物不同层次的作用有复杂的效应和机理,可能对生物分子造成结构的破坏,使其失效。
目前缺少一种成本低廉,且对核酸适用性好的纳米粒制备方法。
发明内容
为解决上述技术问题,本发明提供一种改良型的超声波辅助方法制备核酸纳米粒,使含有核酸的溶液与含有载体化合物的溶液进行迅速混合,同时核酸与载体化合物能够瞬间结合生成纳米粒。本发明旨在提供一种低成本、便捷、重复性好、可放大生产的核酸纳米制剂的制备方法。
本发明的第一个目的是提供了一种核酸纳米粒的制备方法,包括如下步骤:
S1、按重量份计,将1~20份核酸溶解于100~10000份水溶液中,混合均匀得到溶液A,控温备用;
S2、按重量份计,将10~600份载体化合物溶解于100~10000份溶液中,混合均匀得到溶液B,控温备用;
S3、将S1步骤的溶液A至于超声容器中,在超声功率为10~1000W的超声作用下,快速加入溶液B,并继续超声处理10~180s,得到含有核酸纳米粒的溶液;
S4、将含有核酸纳米粒的溶液进行纯化处理,得到所述的核酸纳米粒。
核酸化合物的表面带有大量的磷酸基团,在大多数条件下,表面携带大量的负电荷,能够与携带正电荷的化合物实现静电吸附。若采用带有正电荷的载体化合物与核酸进行快速混合,则能够迅速生成紧实的纳米粒。本发明方法中,将含有正电荷的载体分子溶液,迅速倾倒入含有核酸分子的溶液中,辅以超声震荡,能够实现载体分子和核酸分子两种溶液的瞬间均匀混合。由于两种分子携带不同的电荷,混合后的分子能够迅速结合并发生自组装,形成均匀的纳米粒。其中,快速加入/迅速倾倒在实验室操作中指的是瞬间倾倒。
基于上述方法所形成的纳米粒有如下明显的优点:1.由于超声时间非常短,不会对核酸分子造成破坏;2.由于自组装生成的纳米粒非常紧实,纳米粒中不含有有机溶剂,溶液中的有机溶剂分子可以通过后期简单的纯化步骤分离去除;3.能够适用于水溶性载体,某些载体化合物,如多肽,很难溶于有机溶剂中,因此可以采用该方法将多肽载体分散于水溶液中来制备核酸纳米粒;4.形成的纳米粒大小均匀,通过对条件的筛选能够实现粒径可控、包封率可调,可达到微流控、微射流等大型设备的制备效果。
进一步地,所述的核酸为核糖核酸、脱氧核糖核酸、适配体、核酸类似物中的一种或多种。
进一步地,所述的核糖核酸为信使核糖核酸、转运核糖核酸、端粒酶核糖核酸、反义核糖核酸、小干扰核糖核酸、小激动核糖核酸、微小核糖核酸、小向导核糖核酸、环状核糖核酸、双链核糖核酸、核酶或非编码核糖核酸。
进一步地,所述的水溶液为纯水、缓冲溶液、酸性溶液、碱性溶液、中性溶液中的一种或多种复配水溶液。
进一步地,所述的缓冲溶液为磷酸盐缓冲溶液、柠檬酸盐缓冲溶液、醋酸盐缓冲溶液、三羟甲氨基甲烷缓冲溶液、巴比妥缓冲液、甲酸钠缓冲液或醋酸-锂盐缓冲液。
进一步地,所述的酸性溶液为醋酸溶液、磷酸溶液、柠檬酸溶液、盐酸溶液或草酸溶液。
进一步地,所述的碱性溶液为氨水溶液、三乙胺溶液、三乙醇胺溶液、氢氧化钠溶液或氢氧化钾溶液。
进一步地,所述的中性溶液为氯化钠溶液或氯化钾溶液。
进一步地,所述的载体化合物为中性磷脂、阴离子磷脂、阳离子磷脂、阳离子脂质、阳离子聚合物、阳离子表面活性剂、阳离子多肽、PEG化的脂质、PEG化的磷脂、甾醇中的一种或多种。
进一步地,在S1和S2步骤中,控温是控制温度为0~80℃。
本发明中,温度控制是根据载体性质和所希望形成的纳米粒的性质来共同决定,本领域技术人员可根据工艺条件及技术效果进行常规选择。
进一步地,S2步骤中的溶液为水溶液或有机溶液。
进一步地,所述的水溶液为纯水、缓冲溶液、酸性溶液、碱性溶液、中性溶液中的一种或多种复配水溶液。
进一步地,所述的有机溶液为能与水互溶的有机溶液。
进一步地,所述的有机溶液为甲醇、乙醇、乙二醇、丙二醇、丙三醇、正丙醇、异丙醇、丙酮、丙烯醇、甲醛、乙醛、丙醛、甲酸、乙酸、丙酸、正丁酸、正戊酸、乙二酸、丙二酸、四氢呋喃、N,N-二甲基甲酰胺、二甲基乙酰胺、二甲基亚砜、六甲基磷酰三胺、二氧六环、羟基丙酸、乙胺、乙二胺、吡啶中的一种或多种混合。
本发明的有机溶液包括但不限于上述有机溶剂,但该有机溶液的特点应为能与水互溶。
进一步地,S4步骤中,纯化处理为柱层析、切向流过滤、中空纤维过滤、透析中的一种或多种组合。
进一步地,在S1或S2步骤中,混合是采用超声辅助混合。
借由上述方案,本发明至少具有以下优点:
本发明采用的是改良型的超声方法,在两种液体快速混合的过程中,采用超声波进行辅助混合,使两种溶液能够快速均匀地进行混合,在混合的瞬间,核酸和载体能够快速结合并生成纳米粒,纳米粒的生成时间大约为一分钟左右。生成的纳米粒均匀性好,勿需经过更进一步的均质。该方法反应时间短,纳米粒均匀性高,重复性好,可放大生产。制备的核酸纳米粒可以用作核酸治疗药物,核酸疫苗,核酸农药等。
上述说明仅是本发明技术方案和部分结果的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。
附图说明
图1为本发明实施例1所得到的核酸脂质纳米粒的透射电镜照片;
图2为本发明实施例1所得到的核酸脂质纳米粒的动态光散射数据图;
图3为本发明实施例2所得到的核酸/阳离子聚合物纳米粒的透射电镜照片;
图4为本发明实施例2所得到的核酸/阳离子聚合物纳米粒的动态光散射数据图。
具体实施方式
本发明公开了一种核酸纳米粒的制备方法,本领域技术人员可以借鉴本文内容,适当改进工艺参数实现。特别需要指出的是,所有类似的替换和改动对本领域技术人员来说是显而易见的,它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述,相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合,来实现和应用本发明技术。除非另有定义,本文使用的所有科技术语具有本领域普通技术人员所理解的相同含义。
下面结合实施例,进一步阐述本发明:
实施例1:
所使用的试剂如下:
乙酸-乙酸钠缓冲溶液
二硬脂酰基磷脂酰胆碱
((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯)
胆固醇
小干扰核糖核酸
乙醇
制备工艺:
(a)按重量份数计,将1份小干扰核糖核酸溶解于3000份pH=4.7,0.1M的乙酸-乙酸钠缓冲溶液中,超声功率设定为50W,超声30秒,然后加热到42℃并保持;
(b)按重量份数计,将20份((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯),5份胆固醇,10份二硬脂酰基磷脂酰胆碱,共同作为载体化合物,溶解于1000份乙醇中,超声功率设定为50W,超声120秒,然后加热到42℃并保持;
(c)将步骤a所得到的溶液,置于超声波容器中,在80W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声30秒,即得到小干扰核酸脂质纳米粒溶液。
(d)将步骤c所得到的核酸纳米粒溶液,采用切向流进行分离纯化,即得到所需的核酸纳米粒溶液。
制剂外观:乳白色溶液;稀释10倍可得到淡淡的白色带蓝色乳光的透明溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为95纳米。包封率96%。
实施例2:
所使用的试剂如下:
磷酸二氢钠-磷酸氢二钠缓冲溶液
聚-β氨基酯(Mw≈5000)
双链核糖核酸
二甲基亚砜
制备工艺:
(a):按重量份数计,将1份双链核糖核酸溶解于3000份pH=5.2,0.05M的磷酸二氢钠-磷酸氢二钠缓冲溶液中,超声混合均匀,然后冷却到10℃并保持;
(b):按重量份数计,将30份聚-β氨基酯溶解于500份二甲基亚砜中,超声混合均匀,然后冷却到10℃并保持;
(c):将步骤a所得到的溶液,置于超声波容器中,在100W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声60秒,即得到双链核糖核酸/阳离子聚合物纳米粒溶液。
(d):将步骤c所得到的双链核糖核酸/阳离子聚合物溶液,采用透析进行分离纯化,即得到所需的双链核糖核酸/阳离子聚合物纳米粒溶液。
制剂外观:乳白色溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为430纳米。包封率100%。
实施例3:
所使用的试剂如下:
乙酸-乙酸钠缓冲溶液
二硬脂酰基磷脂酰胆碱
((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯)
胆固醇
小干扰核糖核酸
乙醇
制备工艺:
(a)按重量份数计,将1份小干扰核糖核酸溶解于6000份pH=4.4,0.05M的乙酸-乙酸钠缓冲溶液中,超声功率设定为50W,超声30秒,然后加热到42℃并保持;
(b)按重量份数计,将20份((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯),5份胆固醇,10份二硬脂酰基磷脂酰胆碱,共同作为载体化合物,溶解于2000份乙醇中,超声功率设定为50W,超声120秒,然后加热到42℃并保持;
(c)将步骤a所得到的溶液,置于超声波容器中,在200W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声10秒,即得到小干扰核酸脂质纳米粒溶液。
(d)将步骤c所得到的核酸纳米粒溶液,采用切向流进行分离纯化,即得到所需的核酸纳米粒溶液。
制剂外观:白色带蓝色乳光的透明溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为65纳米。包封率92%。
实施例4:
所使用的试剂如下:
乙酸-乙酸钠缓冲溶液
二硬脂酰基磷脂酰胆碱
((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯)
胆固醇
小干扰核糖核酸
乙醇
制备工艺:
(a)按重量份数计,将1份小干扰核糖核酸溶解于6000份pH=4.4,0.1M的乙酸-乙酸钠缓冲溶液中,超声功率设定为50W,超声30秒,然后加热到42℃并保持;
(b)按重量份数计,将20份((4-羟基丁基)氮杂二基)双(己烷-6,1-二基)双(2-羟基癸酸酯),5份胆固醇,10份二硬脂酰基磷脂酰胆碱,共同作为载体化合物,溶解于2000份乙醇中,超声功率设定为50W,超声120秒,然后加热到42℃并保持;
(c)将步骤a所得到的溶液,置于超声波容器中,在50W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声180秒,即得到小干扰核酸脂质纳米粒溶液。
(d)将步骤c所得到的核酸纳米粒溶液,采用切向流进行分离纯化,即得到所需的核酸纳米粒溶液。
制剂外观:白色带蓝色乳光的透明溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为75纳米。包封率95%。
实施例5:
磷酸二氢钠-磷酸氢二钠缓冲溶液
多肽TAT(YGRKKRRQRRR-NH2)
双链核糖核酸
制备工艺:
(a):按重量份数计,将1份双链核糖核酸溶解于3000份pH=5.0,0.1M的磷酸二氢钠-磷酸氢二钠缓冲溶液中,超声功率设定为50W,超声30秒,然后加热到40℃并保持;
(b):按重量份数计,将20份多肽溶解于3000份pH=5.0,0.1M的磷酸二氢钠-磷酸氢二钠缓冲溶液中,超声功率设定为50W,超声180秒,然后加热到40℃并保持。
(c):将步骤a所得到的溶液,置于超声波容器中,在100W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声60秒,即得到双链核糖核酸/多肽纳米粒溶液。
(d):将步骤c所得到的双链核糖核酸/多肽溶液,采用透析进行分离纯化,即得到所需的双链核糖核酸/多肽纳米粒溶液。
制剂外观:白色带蓝色乳光的透明溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为120纳米。包封率99%。
实施例6:
磷酸二氢钠-磷酸氢二钠缓冲溶液
多肽TAT(YGRKKRRQRRR-NH2)
双链核糖核酸
制备工艺:
(a):按重量份数计,将1份双链核糖核酸溶解于3000份pH=4.8,0.05M的磷酸二氢钠-磷酸氢二钠缓冲溶液中,超声功率设定为50W,超声30秒,然后加热到40℃并保持;
(b):按重量份数计,将20份多肽溶解于3000份pH=5.0,0.1M的磷酸二氢钠-磷酸氢二钠缓冲溶液中,超声功率设定为50W,超声180秒,然后加热到40℃并保持。
(c):将步骤a所得到的溶液,置于超声波容器中,在80W功率的超声波作用下,迅速加入步骤b所得到的溶液,并继续超声120秒,即得到双链核糖核酸/多肽纳米粒溶液。
(d):将步骤c所得到的双链核糖核酸/多肽溶液,采用透析进行分离纯化,即得到所需的双链核糖核酸/多肽纳米粒溶液。
制剂外观:白色带蓝色乳光的透明溶液。
粒度检测方法:动态光散射,透射电镜。
包封率检测方法:Qubit4.0荧光定量检测仪。
检测结果:平均粒径为100纳米。包封率95%。
以上仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。
Claims (10)
1.一种核酸纳米粒的制备方法,其特征在于,包括如下步骤:
S1、按重量份计,将1~20份核酸溶解于100~10000份水溶液中,混合均匀得到溶液A,控温备用;
S2、按重量份计,将10~600份载体化合物溶解于100~10000份溶液中,混合均匀得到溶液B,控温备用;
S3、将S1步骤的溶液A至于超声容器中,在超声功率为10~1000W的超声作用下,快速加入溶液B,并继续超声处理10~180s,得到含有核酸纳米粒的溶液;
S4、将含有核酸纳米粒的溶液进行纯化处理,得到所述的核酸纳米粒。
2.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,所述的核酸为核糖核酸、脱氧核糖核酸、适配体、核酸类似物中的一种或多种。
3.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,所述的水溶液为纯水、缓冲溶液、酸性溶液、碱性溶液、中性溶液中的一种或多种复配水溶液。
4.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,所述的载体化合物为中性磷脂、阴离子磷脂、阳离子磷脂、阳离子脂质、阳离子聚合物、阳离子表面活性剂、阳离子多肽、PEG化的脂质、PEG化的磷脂、甾醇中的一种或多种。
5.根据权利要求4所述的核酸纳米粒的制备方法,其特征在于,在S1和S2步骤中,控温是控制温度为0~80℃。
6.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,S2步骤中的溶液为水溶液或有机溶液。
7.根据权利要求6所述的核酸纳米粒的制备方法,其特征在于,所述的有机溶液为能与水互溶的有机溶液。
8.根据权利要求7所述的核酸纳米粒的制备方法,其特征在于,所述的有机溶液为甲醇、乙醇、乙二醇、丙二醇、丙三醇、正丙醇、异丙醇、丙酮、丙烯醇、甲醛、乙醛、丙醛、甲酸、乙酸、丙酸、正丁酸、正戊酸、乙二酸、丙二酸、四氢呋喃、N,N-二甲基甲酰胺、二甲基乙酰胺、二甲基亚砜、六甲基磷酰三胺、二氧六环、羟基丙酸、乙胺、乙二胺、吡啶中的一种或多种混合。
9.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,S4步骤中,纯化处理为柱层析、切向流过滤、中空纤维过滤、透析中的一种或多种组合。
10.根据权利要求1所述的核酸纳米粒的制备方法,其特征在于,在S1或S2步骤中,混合是采用超声辅助混合。
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