CN115120560B - 一种抗肿瘤靶向药物递送系统及其制备方法和应用 - Google Patents
一种抗肿瘤靶向药物递送系统及其制备方法和应用 Download PDFInfo
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Abstract
一种抗肿瘤靶向药物递送系统包括枸杞多糖、肿瘤抗原CD155质粒、脂质体,枸杞多糖、肿瘤抗原CD155质粒包裹在脂质体内。靶向药物递送系统的制备方法包括以下步骤:步骤S001:构建pcDNA3.1+‑CD155重组载体;步骤S002:制备肿瘤抗原CD155质粒;步骤S003:制备脂质体;步骤S004:用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒,形成脂质体纳米粒,即靶向药物递送系统。本发明的靶向药物递送系统应用于抗肿瘤药物。本发明的靶向药物递送系统具有靶向性、缓释性、降低药物毒性以及提高药物稳定性的效果。
Description
技术领域
本发明涉及靶向药物技术领域,尤其涉及一种抗肿瘤靶向药物递送系统及其制备方法和应用。
背景技术
现有技术的抗肿瘤药物存在的副作用严重、易产生耐药性、药物半衰期短、靶向性差、价格昂贵等问题。
发明内容
有鉴于此,有必要提供一种副作用小、靶向性高、稳定性强的抗肿瘤靶向药物递送系统。
还有必要提供一种抗肿瘤靶向药物递送系统的制备方法。
还有必要提供一种抗肿瘤靶向药物递送系统的应用。
一种抗肿瘤靶向药物递送系统包括
枸杞多糖、肿瘤抗原CD155质粒、脂质体,枸杞多糖、肿瘤抗原CD155质粒包裹在脂质体内。
抗肿瘤靶向药物递送系统的制备方法包括以下步骤:
步骤S001:构建pcDNA3.1+-CD155重组载体;
步骤S002:制备肿瘤抗原CD155质粒;
步骤S003:制备脂质体;
步骤S004:用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒,形成脂质体纳米粒,即抗肿瘤靶向药物递送系统。
一种抗肿瘤抗肿瘤靶向药物递送系统在抗肿瘤药物中的应用。
有益效果:本发明的抗肿瘤靶向药物递送系统相比于现有抗癌药物具有以下效果:
靶向性:抗肿瘤靶向药物递送系统进入体内可被巨噬细胞视为外界异物而吞噬。静脉给予抗肿瘤靶向药物递送系统时,能选择性地集中于单核吞噬细胞系统,防止肿瘤扩散转移。
缓释性:将药物包封于脂质体后,药物会缓慢释放、减缓代谢和排泄,从而延长药物的作用时间。
降低药物毒性:药物被脂质体包封后,主要被网状内皮系统的巨噬细胞所吞噬,集中在肝、脾和骨髓等网状内皮细胞丰富的器官,而相对的,药物在心、肾中累积量明显降低。
提高药物稳定性:不稳定的枸杞多糖被脂质体包封后,受到脂质体双层膜的保护,可提高其稳定性。
附图说明
图1为抗肿瘤靶向药物递送系统透射电子显微镜(TEM)观察结果。
图2为抗肿瘤靶向药物递送系统细胞毒性实验结果。
图3为促进BMDC诱导T细胞活化及对肿瘤细胞HT-29细胞的杀伤效应。
图3中:A.枸杞多糖脂质体促进BMDC诱导T细胞活化及对肿瘤细胞HT-29细胞24h的杀伤效应。B.枸杞多糖脂质体促进BMDC诱导T细胞活化及对肿瘤细胞HT-29细胞48h的杀伤效应,*P<0.05,**P<0.01,***P<0.001,****P<0.0001,n=3。
具体实施方式
为了更清楚地说明本发明实施例的技术方案,下面将对实施例予以说明。
一种抗肿瘤靶向药物递送系统包括枸杞多糖、肿瘤抗原CD155质粒、脂质体,枸杞多糖、肿瘤抗原CD155质粒包裹在脂质体内。
优选的,所述包裹在脂质体内的枸杞多糖、肿瘤抗原CD155质粒的比例为500:1。
抗肿瘤靶向药物递送系统的制备方法包括以下步骤:
步骤S001:构建pcDNA3.1+-CD155重组载体;
步骤S002:制备肿瘤抗原CD155质粒;
步骤S003:制备脂质体;
步骤S004:用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒,形成脂质体纳米粒,即抗肿瘤靶向药物递送系统。
优选的,所述pcDNA3.1+-CD155重组载体的构建包括以下步骤:
步骤S101,设计CD155基因的PCR扩增引物并人工合成得到CD155基因的PCR扩增引物;
设计CD155基因的PCR扩增引物的一较佳实施方式为:从NCBI的Genebank中获取CD155基因(NM_006505.5)编码区序列,选择NheI和HindIII限制性核酸内切酶作为质粒构建的酶切位点,用Snapgene软件设计PCR扩增引物,并送至上海生工生物Sangon Biotech公司合成。
步骤S102,获取总RNA,并进行逆转录,得到与CD155基因的编码区序列的互补DNA,即cDNA;
步骤S103,将PCR扩增引物和cDNA混合进行PCR扩增,得到含有CD155基因的PCR扩增产物;
步骤S104,将pcDNA3.1+载体和PCR扩增产物进行双酶切,得到pcDNA3.1+双酶切产物和PCR双酶切产物;
pcDNA3.1+双酶切产物反应体系如表1所示:
表1:pcDNA3.1+产物酶切反应体系
PCR产物酶切反应体系如表2所示:
表2:PCR产物酶切反应体系
步骤S105,将pcDNA3.1+双酶切产物和PCR双酶切产物进行连接重组,得到pcDNA3.1+-CD155重组载体,具体的,用Takara的T4 DNA Ligase连接酶连接,目的基因与空载体的摩尔比为10:1混合后4℃反应16h,反应体系如表3所示。
表3:酶连反应体系
优选的,步骤101中,上游引物为cgGCTAGCATGGCCCGAGCCATGG,下游引物为ccAAGCTTTCACCTTGTGCCCTCTGTCTG,上游引物含有酶切位点NheI,下游引物含有酶切位点HindIII。
优选的,步骤S102中,总RNA的获取过程为:
a.培养HT-29细胞,当HT-29细胞长至80%~90%时,收集HT-29细胞,置于1.5mLRNase-free离心管中,每管加1mL裂解液,室温放置5min;
b.在RNase-free离心管中加入200μL的氯仿,混匀后在涡旋仪器上进行涡旋15s,之后在室温下静置3min;
c.将RNase-free离心管在12000rpm的转速下离心10min,之后将水相转移到第二个RNase-free离心管中;
d.在第二个RNase-free离心管中加入0.5倍体积的无水乙醇,之后将RNase-free离心管中的物质转入到吸附柱中,之后在4℃的温度下,将吸附柱12000rpm离心30s;
e.在吸附柱加入500μL RD,在12000rpm的转速下离心30s,之后将吸附柱内的液体去掉;
f.加入500μLRW到吸附柱中,室温静置2min,12000rpm离心30s,之后将吸附柱内的液体去掉;
g.将吸附柱转移至第三个RNase-free离心管中,在4℃的温度下,12000rpm离心2min;
h.将吸附柱转移至第四个RNase-free离心管中,加入30μL RNase-free水,室温静置2min,12000rpm离心2min,收集总RNA。
优选的,步骤S102中,逆转录的过程为:
将总RNA和通用引物充分混匀,在PCR仪中65℃孵育5min,混匀并离心。如表4,加入反应缓冲液、核糖核酸酶抑制剂、逆转录酶、三磷酸脱氧核苷酸混合溶液,再置于PCR仪进行逆转录,得到cDNA。
表4:逆转录反应体系
优选的,步骤S103的具体过程为:
如表5所示,将2×TransTaqHiFi PCR SuperMixI(-dye)、cDNA、上游引物、下游引物、RNase-free水混合在一起,在PCR仪内经过
预变性:94℃,4min;
变性:94℃,30s;
退火:56℃,30s;
延伸:72℃,1min;
如此经历30个循环;
之后总延伸:72℃,5min,得到含有CD155基因的PCR扩增产物,之后通过琼脂糖凝胶对PCR扩增产物进行纯化。
表5:PCR反应体系
成分 | 用量(μL) |
2×TransTaqHiFi PCR SuperMixI(-dye) | 25 |
cDNA | 10 |
上游引物 | 1 |
下游引物 | 1 |
RNase-free Water | 13 |
优选的,所述制备肿瘤抗原CD155质粒包括以下步骤:
步骤S201,将pcDNA3.1+-CD155重组载体加入100μL大肠杆菌DH5α菌液,冰浴30min,之后42℃热激90s,移回至冰上,冰浴5min,得到含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α菌液;
步骤S202,将含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α移入至500μL无菌的不含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养1h;
步骤S303,取100μL经过振荡培养的大肠杆菌DH5α菌液接种到含Amp的LB培养基上,在37℃的环境下培养18~24h,得到含单克隆菌落的培养液;
步骤S304,取5个单克隆菌落接种于3mL的含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养12~16h;
步骤S305,收集菌液,12000rpm离心1min,弃掉上清液,在剩下的物质中提取CD155质粒。
优选的,用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒的步骤为:
步骤S401,将枸杞多糖和CD155质粒溶于PBS缓冲液中作为水相,在40摄氏度的温度下进行水浴,并通过旋蒸减压蒸发以将水相中的有机溶剂除去;
步骤S402,当水相物质旋蒸呈胶状物时加入6mLPBS缓冲液,继续旋蒸1h直至完全除去有机溶剂,得到包覆枸杞多糖和肿瘤抗原CD155质粒的脂质体悬液;
步骤S403,将脂质体悬液通过0.22μm的滤菌器除菌,得到脂质体纳米粒。
通过TEM观察脂质体的形态特征,如图1所示,在2500倍、5000倍以及20000倍的放大倍数下,均可观察到脂质体纳米粒能够形成尺寸、形态较为均一的球形颗粒。
为了验证本发明的抗肿瘤靶向药物递送系统的实施效果,以下将结合实验予以说明。
其中涉及到的实验组有:空白脂质体组(L)、枸杞多糖脂质体组(LBPL)、CD155质粒脂质体组(CD155L)、靶向药物系统组(LBP-CD155L)。
通过马尔文激光粒度分析仪测量脂质体的粒径,空白脂质体、枸杞多糖脂质体、CD155质粒脂质体、靶向药物系统的平均粒径分别为177.7±78.15nm、128.4±47.29nm、133.1±9.211nm和224.4±17.09nm;多分散系数(PDI)分别为0.198、0.193、0.005、0.006,均小于0.3,符合脂质体稳定性要求。在4摄氏度的储藏温度下,各组在第1、7、14和90天的粒径和PDI变化如表6所示,粒径和PDI变化较小,说明制备出的脂质体稳定性好,不容易聚集。
表6:各组储藏稳定性实验结果
通过CCK 8试剂盒对各组进行细胞毒性实验,结果如图2所示,各组在浓度在最大浓度500μg/mL时细胞的活力均大于1,说明制备出的脂质体对细胞无毒性。
以下将通过对比枸杞多糖脂质体组(LBPL)、CD155质粒脂质体组(CD155L)、靶向药物系统组(LBP-CD155L)对肿瘤细胞HT-29(人结肠癌细胞)的杀伤效应,来验证本发明的靶向药物系统的效果。
将肿瘤细胞HT-29与活化的CD8+T细胞按1:5的数量比培养,分别在24h和48h收集所有细胞,经FITC Annexin V Apoptosis Detection Kit I染色后,FCM检测其凋亡率。
培养24小时和培养48小时的结果如图3所示,靶向药物系统与其它组相比,肿瘤细胞HT-29的凋亡率明显升高,其中LBP-CD155L与CD155L相比,24小时的结果P<0.01,48小时的结果P<0.0001;LBP-CD155L与LBPL相比,24小时的结果P<0.05,48小时的结果P<0.0001,说明枸杞多糖与CD155质粒组合形成的脂质体,即靶向药物系统相比于枸杞多糖或CD155质粒的脂质体,对肿瘤细胞HT-29具有显著的杀伤效果。
因此,可以说明抗肿瘤靶向药物递送系统能够应用于抗肿瘤药物。
以上所揭露的仅为本发明较佳实施例而已,当然不能以此来限定本发明之权利范围,本领域普通技术人员可以理解实现上述实施例的全部或部分流程,并依本发明权利要求所作的等同变化,仍属于发明所涵盖的范围。
Claims (6)
1.一种抗肿瘤靶向药物递送系统,其特征在于:所述抗肿瘤靶向药物递送系统由枸杞多糖、肿瘤抗原CD155质粒、脂质体组成,枸杞多糖、肿瘤抗原CD155质粒包裹在脂质体内;
所述包裹在脂质体内的枸杞多糖、肿瘤抗原CD155质粒的比例为500:1;
上述抗肿瘤靶向药物递送系统采用如下制备方法获得:
步骤S001:构建pcDNA3.1 + -CD155重组载体;
步骤S002:制备肿瘤抗原CD155质粒;
步骤S003:制备脂质体;
步骤S004:用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒,形成脂质体纳米粒,即抗肿瘤靶向药物递送系统;
步骤“步骤S001”的所述pcDNA3.1 + -CD155重组载体的构建包括以下步骤:
步骤S101,设计CD155基因的PCR扩增引物并人工合成得到CD155基因的PCR扩增引物;
步骤S102,获取总RNA,并进行逆转录,得到与CD155基因的编码区序列的互补DNA,即cDNA;
步骤S103,将PCR扩增引物和cDNA混合进行PCR扩增,得到含有CD155基因的PCR扩增产物;
步骤S104,将pcDNA3.1 + 载体和PCR扩增产物进行双酶切,得到pcDNA3.1 + 双酶切产物和PCR双酶切产物;
步骤S105,将pcDNA3.1 + 双酶切产物和PCR双酶切产物进行连接重组,得到pcDNA3.1+ -CD155重组载体;
所述步骤S101中,上游引物为cgGCTAGCATGGCCCGAGCCATGG,下游引物为ccAAGCTTTCACCTTGTGCCCTCTGTCTG,上游引物含有酶切位点 Nhe I ,下游引物含有酶切位点 Hind III;
其中,所述制备肿瘤抗原CD155质粒包括以下步骤:
步骤S201,将pcDNA3.1+-CD155重组载体加入100μL大肠杆菌DH5α菌液,冰浴30min,之后42℃热激90s,移回至冰上,冰浴5min,得到含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α菌液;
步骤S202,将含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α移入至500μL无菌的不含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养1h;
步骤S303,取100μL经过振荡培养的大肠杆菌DH5α菌液接种到含Amp的LB培养基上,在37℃的环境下培养18~24h,得到含单克隆菌落的培养液;
步骤S304,取5个单克隆菌落接种于3mL的含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养12~16h;
步骤S305,收集菌液,12000rpm离心1min,弃掉上清液,在剩下的物质中提取CD155质粒。
2.一种如权利要求1所述的抗肿瘤靶向药物递送系统的制备方法,其特征在于:
步骤S001:构建pcDNA3.1 + -CD155重组载体;
步骤S002:制备肿瘤抗原CD155质粒;
步骤S003:制备脂质体;
步骤S004:用脂质体包覆枸杞多糖和肿瘤抗原CD155质粒,形成脂质体纳米粒,即抗肿瘤靶向药物递送系统;
步骤“步骤S001”的所述pcDNA3.1 + -CD155重组载体的构建包括以下步骤:
步骤S101,设计CD155基因的PCR扩增引物并人工合成得到CD155基因的PCR扩增引物;
步骤S102,获取总RNA,并进行逆转录,得到与CD155基因的编码区序列的互补DNA,即cDNA;
步骤S103,将PCR扩增引物和cDNA混合进行PCR扩增,得到含有CD155基因的PCR扩增产物;
步骤S104,将pcDNA3.1 + 载体和PCR扩增产物进行双酶切,得到pcDNA3.1 + 双酶切产物和PCR双酶切产物;
步骤S105,将pcDNA3.1 + 双酶切产物和PCR双酶切产物进行连接重组,得到pcDNA3.1+ -CD155重组载体;
所述步骤S101中,上游引物为cgGCTAGCATGGCCCGAGCCATGG,下游引物为ccAAGCTTTCACCTTGTGCCCTCTGTCTG,上游引物含有酶切位点 Nhe I ,下游引物含有酶切位点 Hind III;
其中,所述制备肿瘤抗原CD155质粒包括以下步骤:
步骤S201,将pcDNA3.1+-CD155重组载体加入100μL大肠杆菌DH5α菌液,冰浴30min,之后42℃热激90s,移回至冰上,冰浴5min,得到含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α菌液;
步骤S202,将含有pcDNA3.1+-CD155重组载体的大肠杆菌DH5α移入至500μL无菌的不含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养1h;
步骤S303,取100μL经过振荡培养的大肠杆菌DH5α菌液接种到含Amp的LB培养基上,在37℃的环境下培养18~24h,得到含单克隆菌落的培养液;
步骤S304,取5个单克隆菌落接种于3mL的含Amp的LB培养基上,在37℃的环境下,以200rpm的转速振荡培养12~16h;
步骤S305,收集菌液,12000rpm离心1min,弃掉上清液,在剩下的物质中提取CD155质粒。
3.如权利要求2所述的抗肿瘤靶向药物递送系统的制备方法,其特征在于:步骤S102中,总RNA的获取过程为:
a. 培养HT-29细胞,当HT-29细胞长至80%~90%时,收集HT-29细胞,置于1.5 mLRNase-free离心管中,每管加1mL裂解液,室温放置5 min;
b. 在RNase-free离心管中加入200 μL的氯仿,混匀后在涡旋仪器上进行涡旋15 s ,之后在室温下静置3 min;
c.将RNase-free离心管在12000 rpm的转速下离心10 min,之后将水相转移到第二个RNase-free离心管中;
d. 在第二个RNase-free离心管中加入0.5倍体积的无水乙醇,之后将RNase-free离心管中的物质转入到吸附柱中,之后在4℃的温度下,将吸附柱12000 rpm离心30 s;
e. 在吸附柱加入500 μL RD,在12000 rpm的转速下离心30 s,之后将吸附柱内的液体去掉;
f. 加入500 μLRW到吸附柱中,室温静置2 min,12000 rpm离心30 s,之后将吸附柱内的液体去掉;
g. 将吸附柱转移至第三个RNase-free离心管中,在4℃的温度下,12000 rpm离心2min;
h. 将吸附柱转移至第四个RNase-free离心管中,加入30 μL RNase-free水,室温静置2 min,12000 rpm离心2 min,收集总RNA。
4.如权利要求3所述的抗肿瘤靶向药物递送系统的制备方法,其特征在于:步骤S102中,逆转录的过程为:
将总RNA和通用引物充分混匀,在PCR仪中65℃孵育5 min,混匀并离心加入反应缓冲液、核糖核酸酶抑制剂、逆转录酶、三磷酸脱氧核苷酸混合溶液,再置于PCR仪进行逆转录,得到cDNA。
5.如权利要求4所述的抗肿瘤靶向药物递送系统的制备方法,其特征在于:步骤S103的具体过程为:
将2×TransTaqHiFi PCR SuperMixI(-dye)、cDNA、上游引物、下游引物、RNase-free水混合在一起,在PCR仪内经过
预变性:94 ℃ ,4 min;
变性:94 ℃,30s;
退火:56 ℃,30s;
延伸:72 ℃,1min;
如此经历30个循环;
之后总延伸:72 ℃,5min,得到含有CD155基因的PCR扩增产物,之后通过琼脂糖凝胶对PCR扩增产物进行纯化。
6.一种如权利要求1所述的抗肿瘤靶向药物递送系统在制备抗肿瘤药物中的应用。
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