CN114533888A - 一种以细胞为载体的仿生载药系统及其制备方法和应用 - Google Patents
一种以细胞为载体的仿生载药系统及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种以细胞为载体的仿生载药系统及其制备方法和应用,属于生物材料领域。该制备方法包括先在活的细胞中装载活的微藻,获得具有光合作用能力的细胞;然后在具有光合作用能力的细胞中装载药物、或微米或纳米粒子、或载药粒子,获得以细胞为载体的仿生载药系统。本发明中以细胞为载体的仿生载药系统可以根据需求实现具有靶向肿瘤组织或炎症部位及其乏氧区域的能力和光合作用产氧能力、以及光动力治疗或声动力治疗能力、或发挥所装载的药物或微纳米粒子作用的能力,作为治疗肿瘤或炎症的靶向药物时,可以有效提高对肿瘤和炎症治疗的效率。
Description
技术领域
本发明属于生物材料领域,特别涉及一种以细胞为载体的仿生载药系统及其制备方法和应用。
背景技术
纳米粒子或载药纳米粒子经静脉注射给药后,在体内递送的途中,会被免疫系统识别和清除,导致不到1%的纳米粒子能成功进入肿瘤组织,这是纳米粒子靶向病灶(如肿瘤和炎症)部位效果差的重要原因。因此,制备仿生粒子以特洛伊木马的策略递送药物是提高药物在病灶部位靶向聚集的有效手段,其中以细胞作为递送载体是制备仿生粒子的重要方向。
自体细胞如神经干细胞、间充质干细胞、单核细胞、巨噬细胞等可通过其表面的某些蛋白来逃避免疫系统清除,可以向肿瘤组织或炎症部位迁移,粘附肿瘤细胞,并能进入乏氧区域,特别是巨噬细胞,由于能从患者外周血和腹腔中大量分离,且能装载数量可观的药物和纳米粒子,在药物和纳米粒子递送领域有广阔的应用前景。
肿瘤和炎症组织内氧的分压低,乏氧不仅促进肿瘤侵袭和转移,还降低光敏剂药物产生单线态氧的能力,向肿瘤或炎症组织供氧有重要意义。供氧的方式主要有高压氧疗法,或通过氟碳化合物、金属有机框架、血红蛋白运载氧气的方式向肿瘤供氧,或者通过MnO2纳米粒子以及多价态的金属化合物与瘤内H2O2反应产生氧气、CaO2纳米粒子水解产生氧气等方式向肿瘤供氧,而利用天然微藻的光合作用向肿瘤组织供氧更受研究者青睐[YueQiao,Fei Yang,Tingting Xie,Zhen Du,Danni Zhong,Yuchen Qi,Yangyang Li,WanlinLi,Zhimin Lu,Jianghong Rao,Yi Sun,Min Zhou.Engineered algae:A novel oxygen-generating system for effective treatment of hypoxic cancer.Sci.Adv.2020;6:eaba5996],而如何将微藻高效递送到肿瘤组织,是充分利用微藻的光合作用向肿瘤供氧的关键,以细胞作为载体向肿瘤或炎症部位输送微藻以及同时输送微藻和药物等其它材料未见文献报道。
发明内容
本发明的主要目的是提供一种以细胞为载体的仿生载药系统的制备方法,先以细胞为载体装载微藻获得可通过光合作用产氧的细胞,再在装载有微藻的细胞中进一步装载药物和/或功能材料,提高对肿瘤和炎症治疗的效率。
本发明的另一目的是提供通过上述制备方法得到的以细胞为载体的仿生载药系统。
本发明的再一目的是提供上述以细胞为载体的仿生载药系统在作为治疗肿瘤或炎症的靶向药物中的应用。
为实现上述目的,本发明采用如下技术方案:
本发明提供的以细胞为载体的仿生载药系统的制备方法,包括以下步骤:
(1)在活的细胞中装载活的微藻,获得具有光合作用能力的细胞;
(2)在上述具有光合作用能力的细胞中装载药物、或微米或纳米粒子、或载药粒子,获得以细胞为载体的仿生载药系统。
作为优选,步骤(1)中,具体方法是:先分别培养细胞和微藻,除去细胞和微藻中的培养基,向细胞中加入分散于磷酸盐缓冲液(PBS)或不含血清的细胞培养液中的微藻,且细胞与微藻的个数比为1:1–9,于培养箱中继续培养1–5h,除去细胞中的溶液,用PBS或不含血清的细胞培养液洗涤细胞,即得具有光合作用能力的细胞。
作为更优选,步骤(1)中,加入细胞与微藻的个数比为1:1–5。
作为优选,步骤(2)中,具体方法是:先按与上述步骤(1)相同方法制备获得具有光合作用能力的细胞,然后加入分散于PBS或不含血清的细胞培养液中的药物、微米或纳米粒子、或载药粒子,所述具有光合作用能力的细胞与药物的比例满足:当细胞数量为1×105个时,加入的药物为1–20μg;所述具有光合作用能力的细胞与微米或纳米粒子或载药粒子的比例满足:当细胞数量为1×105个时,加入的微米或纳米粒子或载药粒子质量为25–100μg;于培养箱中继续培养1–5h,除去细胞中的溶液,用PBS或不含血清的细胞培养液洗涤细胞,即得以细胞为载体的仿生载药系统。
作为优选,所述活的细胞来源于人或动物(体外)的干细胞或具有吞噬功能的细胞,进一步是单核细胞或巨噬细胞。
作为优选,所述微藻选自蓝藻门、绿藻门、金藻门和红藻门中的任意一种微藻。
作为更优选,所述微藻为蛋白核小球藻GY-D19。
作为优选,所述药物选自光敏剂、化疗药物、基因药物中的一种或两种以上组合。
作为优选,所述微米或纳米粒子选自磁性氧化铁、替代型磁性铁氧体MFe2O4(M=Mn、Mg、Zn、Co、Fe、Al、Cr)、非磁性氧化铁、二氧化硅、无机半导体粒子、金粒子、银粒子、铂粒子、羟基磷灰石、碳酸钙、过氧化钙、粘土粒子、石墨烯基粒子、活性炭、钻石、聚合物粒子、脂质体中的一种或两种以上组合。
本发明还提供一种以细胞为载体的仿生载药系统,通过上述以细胞为载体的仿生载药系统的制备方法得到。
本发明还提供上述以细胞为载体的仿生载药系统在制备/作为治疗肿瘤或炎症的靶向药物中的应用。
与现有技术相比,本发明的有益效果在于:本发明中以细胞为载体的仿生载药系统可以根据需求实现具有靶向肿瘤组织或炎症部位及其乏氧区域的能力、或光合作用产氧的能力、或光动力治疗或声动力治疗的能力、或发挥所装载的药物或微纳米粒子作用的能力,在制备/作为治疗肿瘤或炎症的靶向药物时,可以有效提高对肿瘤和炎症治疗的效率。
附图说明
图1是巨噬细胞与小球藻按个数比为1:1、1:3、1:5制备得到的载小球藻的巨噬细胞混悬液,MΦ和Chl分别是指巨噬细胞和小球藻。
图2是内吞小球藻的巨噬细胞RAW264.7在激光共聚焦显微镜下的荧光照片,其中巨噬细胞与小球藻按数量比为1:3混合,灰白色箭头所指的灰色球形粒子为小球藻。
图3是内吞小球藻的巨噬细胞RAW264.7在激光共聚焦显微镜下的白光照片,其中巨噬细胞与小球藻按数量比为1:3混合,灰色箭头所指的黑色球形粒子为小球藻。
图4是装载光敏剂Ce6的二氧化硅纳米粒子(Ce6-SiO2)透射电镜照片。
图5是装载SiO2-Ce6的巨噬细胞在激光共聚焦荧光显微镜下拍摄的荧光照片。
图6是同时转载装载小球藻和SiO2-Ce6的巨噬细胞在激光共聚焦荧光显微镜下拍摄的荧光照片。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面结合本发明实施例的附图,对本发明实施例的技术方案进行清楚、完整地描述。显然,所描述的实施例是本发明的一部分实施例,而不是全部的实施例。基于所描述的本发明的实施例,本领域普通技术人员在无需创造性劳动的前提下所获得的所有其它实施例,都属于本发明保护的范围。
实施例1
制备装载小球藻的巨噬细胞,分3个步骤:
培养巨噬细胞RAW264.7:于37℃二氧化碳培养箱中培养,培养基为含10%FBS的DMEM。
培养小球藻:在25℃的光照培养箱中培养蛋白核小球藻GY-D19。
在巨噬细胞中装载小球藻:将巨噬细胞RAW264.7传代至96孔板中,当每孔细胞数量为1×105个时,移去培养基,洗涤细胞,然后向细胞中加入50μL浓度分别为0.2×107、0.6×107、1.0×107、1.4×107、1.8×107个/mL的分散于无血清的DMEM培养基中的小球藻,使混合物中巨噬细胞与小球藻的个数比为1:1、1:3、1:5、1:7、1:9,每个比例有4个复孔,于37℃二氧化碳培养箱中培养2h后,移去培养基,用PBS小心洗涤,然后加入含血清的培养基,于37℃二氧化碳培养箱中培养12h,最后定量检测巨噬细胞成活率。
结果表明,内吞小球藻后,巨噬细胞的颜色由原来的近无色转变为深色,如图1所示。当巨噬细胞与小球藻混合时二者(巨噬细胞:小球藻)的个数比为1:1、1:3、1:5时,巨噬细胞成活率分别为131.1±3.0%、124.1±5.2%、97.4±10.1%,当二者比例为1:7和1:9时,巨噬细胞活性有明显下降,但仍保持在约70%或以上。可见,巨噬细胞能装载数量可观的小球藻,装载后可保持高的活性。
实施例2
观察装载小球藻的巨噬细胞中小球藻的位置以及巨噬细胞形态,方法是:将洗净灭菌的盖玻片置于培养皿中,将巨噬细胞RAW264.7培养于盖玻片上,当细胞数量达3×106个时,移去培养基,洗涤细胞,然后向培养皿中加入2mL浓度为4.5×106个/mL的分散于无血清DMEM培养基中的小球藻,此时混合物中巨噬细胞与小球藻的个数比为1:3。于37℃二氧化碳培养箱中培养2h后,移去培养基,在激光共聚焦荧光显微镜下观察小球藻被巨噬细胞内吞的情况。
结果表明,巨噬细胞中有明亮红色荧光的微球即小球藻,小球藻分布于巨噬细胞的细胞质中,所加入的小球藻在2h内几乎全被巨噬细胞所内吞,巨噬细胞形态有所增大,并伸出触角,如图2和3所示。
实施例3
巨噬细胞培养方式与实施例1相同,在96孔板中,巨噬细胞与小球藻的个数比为1:3。当向巨噬细胞中加完小球藻后,于37℃二氧化碳培养箱中分别培养1h和5h。
结果表明,巨噬细胞均保持高的活性,共培养1h的巨噬细胞,内吞小球藻的数量比共培养5h的巨噬细胞要少,共培养5h的巨噬细胞内吞小球藻的数量高于共培养2h的巨噬细胞内吞的量。
实施例4
制备载药二氧化硅,方法是:在直径为100nm左右的二氧化硅表面修饰3-氨丙基三乙氧基硅烷,使二氧化硅表面带氨基,然后通过羧基活化试剂1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐和N-羟基琥珀酰亚胺将光敏剂药物二氢卟吩e6(Ce6)连接到二氧化硅(SiO2)表面,同时,更多的Ce6通过π-π键的堆叠等作用结合到反应在SiO2表面的Ce6上。结果可见,得到载药量为13.0±0.4%的载药二氧化硅纳米粒子(即Ce6-SiO2),Ce6-SiO2保持SiO2原有的球形形貌(图4),在荧光显微镜下发射明亮的红色荧光。
将Ce6-SiO2(其中Ce6浓度:100μg/mL)加入到巨噬细胞中,培养3h后,在激光共聚焦荧光显微镜下可见,细胞呈现明亮的红色荧光,这些红色荧光主要分布在巨噬细胞的细胞质中(图5)。
按实施例2制备好装载小球藻的巨噬细胞,移去培养基,PBS小心洗涤,96孔板中每孔中的巨噬细胞数量维持在1×105个,然后向细胞中加入用不含血清的细胞培养基DMEM分散的Ce6-SiO2,其中Ce6的浓度分别为50、100、200μg/mL,于37℃二氧化碳培养箱中培养1–5h。用不含血清的DMEM洗涤细胞,加入含血清的DMEM,于37℃二氧化碳培养箱中继续培养12h,检测巨噬细胞成活率,实验重复4次。
结果表明,巨噬细胞能同时装载小球藻和Ce6-SiO2,在荧光显微镜下观察可见,小球藻和Ce6-SiO2均分布在巨噬细胞的细胞质中(图6)。当Ce6-SiO2中的Ce6的浓度分别为50、100、200μg/mL,与装载小球藻的巨噬细胞共培养3h时,巨噬细胞成活率分别为93.5±4.7%、80.5±2.3%、81.7±5.5%,即细胞保持较高的活性;当共培养1h时,巨噬细胞活性与上述相比有所提高;当共培养5h时,巨噬细胞活性与上述相比有所下降。
以上所述为本发明的较佳实施例而已,但本发明不应该局限于该实施例所公开的内容。所以凡是不脱离本发明所公开的精神下完成的等效或修改,都落入本发明保护的范围。
Claims (10)
1.一种以细胞为载体的仿生载药系统的制备方法,其特征在于,包括以下步骤:
(1)在活的细胞中装载活的微藻,获得具有光合作用能力的细胞;
(2)在上述具有光合作用能力的细胞中装载药物、或装载微米或纳米粒子、或装载载药粒子,获得以细胞为载体的仿生载药系统。
2.根据权利要求1所述以细胞为载体的仿生载药系统的制备方法,其特征在于,步骤(1)中,方法是:先分别培养细胞和微藻,除去细胞和微藻中的培养基,向细胞中加入分散于磷酸盐缓冲液(PBS)或分散于不含血清的细胞培养液中的微藻,且细胞与微藻的个数比为1:1–9;于培养箱中继续培养1–5h,除去细胞中的溶液,用PBS或不含血清的细胞培养液洗涤细胞,即得具有光合作用能力的细胞。
3.根据权利要求1或2所述以细胞为载体的仿生载药系统的制备方法,其特征在于,步骤(2)中,方法是:向所述具有光合作用能力的细胞加入分散于PBS或分散于不含血清的细胞培养液中的药物、或微米或纳米粒子、或载药粒子,所述具有光合作用能力的细胞与药物的比例满足:当细胞数量为1×105个时,加入的药物为1–20μg;所述具有光合作用能力的细胞与微米或纳米粒子或载药粒子的比例满足:当细胞数量为1×105个时,加入的微米或纳米粒子或载药粒子质量为25–100μg;于培养箱中继续培养1–5h,除去细胞中的溶液,用PBS或不含血清的细胞培养液洗涤细胞,即得以细胞为载体的仿生载药系统。
4.根据权利要求1所述以细胞为载体的仿生载药系统的制备方法,其特征在于,所述活的细胞来源于人或动物的干细胞或具有吞噬功能的细胞。
5.根据权利要求4所述以细胞为载体的仿生载药系统的制备方法,其特征在于,所述活的细胞为单核细胞或巨噬细胞。
6.根据权利要求1所述以细胞为载体的仿生载药系统的制备方法,其特征在于,所述微藻选自蓝藻门、绿藻门、金藻门和红藻门中的任意一种微藻。
7.根据权利要求6所述以细胞为载体的仿生载药系统的制备方法,其特征在于,所述微藻为蛋白核小球藻。
8.根据权利要求1所述以细胞为载体的仿生载药系统的制备方法,其特征在于,所述药物选自光敏剂、化疗药物、基因药物中的一种或两种以上组合;
所述微米或纳米粒子选自磁性氧化铁、替代型磁性铁氧体MFe2O4(M选自Mn、Mg、Zn、Co、Fe、Al、Cr)、非磁性氧化铁、二氧化硅、无机半导体粒子、金粒子、银粒子、铂粒子、羟基磷灰石、碳酸钙、过氧化钙、粘土粒子、石墨烯基粒子、活性炭、钻石、聚合物粒子、脂质体中的一种或两种以上组合。
9.一种以细胞为载体的仿生载药系统,通过权利要求1至8任一项所述以细胞为载体的仿生载药系统的制备方法得到。
10.权利要求9所述以细胞为载体的仿生载药系统用作治疗肿瘤或炎症的靶向药物。
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