CN110115765B - 兼具分子靶向/基因/光热治疗的纳米复合物的制备方法 - Google Patents
兼具分子靶向/基因/光热治疗的纳米复合物的制备方法 Download PDFInfo
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Abstract
本发明属于生物医药领域,涉及兼具分子靶向/基因/光热治疗的纳米复合物的制备方法。该纳米复合物是通过将壳聚糖衍生物CE7Q和CQ混合后运载Survivin shRNA的表达质粒SV得到的CE7Q/CQ/S。本发明利用连接在CE7Q壳聚糖衍生物上的厄洛替尼实现对EGFR突变型肺癌的特异性识别;通过连接CE7Q壳聚糖衍生物上的荧光分子Cy7进行近红外荧光成像和光热治疗;用经过季铵盐修饰的CQ壳聚糖衍生物和CE7Q混合后运载基因药物SV下调Survivin的表达,联合分子靶向/基因/光热治疗实现EGFR突变型肺癌的高效治疗,逆转EGFR‑TKIs耐药。
Description
技术领域
本发明属于生物医药领域,具体涉及兼具分子靶向/基因/光热治疗的纳米复合物的制备方法。
背景技术
癌症的发生是多种因素共同作用的结果,其生物学的复杂性导致单药治疗经常不能满足临床需求,并且单药的一维作用机制经常会激活或加强替代途径,促使化疗耐药,甚至导致肿瘤复发的出现。表皮生长因子受体酪氨酸激酶抑制剂(EGFR-TKIs)在对EGFR突变的非小细胞肺癌(NSCLC)的治疗初期具有优越的治疗效果,然后在接受EGFR-TKIs治疗一段时间后,许多患者会出现耐药(Huang L , Fu L . Mechanisms of resistance to EGFRtyrosine kinase inhibitors[J]. Acta Pharmaceutica Sinica B, 2015, 5(5):S2211383515001045.)。EGFR-TKIs的耐药与信号传导中有关因子的表达异常、DNA 修复异常和凋亡基因的异常表达等存在密切关系。凋亡蛋白抑制因子Survivin在肺癌中呈现高表达,并且过量表达的Survivin参与EGFR-TKIs的耐药。研究人员发现,通过基因沉默下调Survivin的表达可以提高耐药细胞对EGFR-TKIs的敏感性(Shi W L , Li J , Bao Q L ,et al. Survivin mRNA expression in blood as a predictor of the response toEGFR-tyrosine kinase inhibitors and prognosis in patients with non-small celllung cancer[J]. Medical Oncology, 2014, 31(4):893-1791.)。
近年来,光热治疗( PTT)作为一种微创的肿瘤治疗方式已被广泛研究。 PTT是一种依靠光热剂在近红外(NIR)光照射下产生足够的热量来抑制肿瘤生长甚至消融肿瘤的手段。与手术、化疗、放疗等传统肿瘤治疗模式相比,PTT具有侵袭性小、副作用少、特异性高等优点。大量临床试验表明,PTT可增强化疗疗效,已作为一种增敏手段应用于肿瘤的联合治疗。在NIR光照下,七甲川菁染料Cy7可以通过吸收近红外光,将能量转化为局部热量进行光热治疗。
通过适当的药物组合,达到多种治疗方式的协同抗肿瘤效果,已成为治疗耐药癌症的主要策略。药物输送系统能够通过载体将多种类型的治疗药物或诊断剂递送到目标部位,并且减小对正常组织的伤害。壳聚糖作为一种多用途的生物多糖,带有高正电荷,可以与带负电荷的DNA形成稳定的复合物,并将DNA递送到细胞内表达目的基因。壳聚糖骨架上的-OH和-NH2基团易于修饰,通过偶联药物或靶标,构建多功能纳米载药系统成为研究热点(Prabaharan, M. Chitosan-based nanoparticles for tumor-targeted drug delivery[J]. International Journal of Biological Macromolecules, 2015, 72:1313-1322.)。
因此,我们构建一种能共输送分子靶向药物、基因药物和光热剂的纳米复合物,既可以用于近红外荧光成像,又能实现EGFR突变型NSCLC的高效治疗,逆转EGFR-TKIs的耐药。
发明内容
本发明的目的在于提供一种兼具分子靶向/基因/光热治疗的纳米复合物的制备方法。制备所得纳米复合物既能够通过近红外荧光成像诊断不同肺癌的分子分型,又能实现EGFR突变型肺癌的高效治疗。
为达到上述目的,本发明采取了如下技术方案:
一种兼具分子靶向/基因/光热治疗的纳米复合物,它是通过在壳聚糖骨架上偶联分子靶向药物厄洛替尼Er、荧光分子Cy7和炔丙基季铵溴盐Q-amine得到CE7Q,CE7Q与季铵盐修饰的壳聚糖CQ混合后,共负载基因药物Survivin短发夹RNA(shRNA)的表达质粒SV,得到纳米复合物CE7Q/CQ/S。CE7Q、CQ和SV的质量比为0.5:1:0.1~5:1:3。
Cy7的合成步骤依据文献:Yang Z, Lee J H, Jeon H M, et al. Folate-BasedNear-Infrared Fluorescent TheranosticGemcitabine Delivery[J]. Journal of theAmerican Chemical Society, 2013, 135(31):11657-11662. 。
炔丙基季铵溴Q-amine的合成步骤依据文献:Nguyen, H. K.; Fournier, O.;Asseline, U.; Dupret, D.; Thuong, N. T. Nucleic Acids Res. 1999, 27, 1492–1498.。
CQ的合成步骤依据文献:Gao Y, Zhang Z, Chen L, Gu W, Li Y. Synthesis of6-N,N,N-trimethyltriazole chitosan via "click chemistry" and evaluation forgene delivery. Biomacromolecules. 2009, 10(8):2175-82.。
一种兼具分子靶向/基因/光热治疗的纳米复合物的制备方法,包括以下步骤:
(1)用ddH2O分别将CE7Q 、CQ和SV配置成溶液;
(2)然后按质量比将CE7Q溶液 、CQ溶液混合均匀后得到CE7Q/CQ溶液;
(3)在涡旋状态下按质量比将SV溶液慢慢滴加到步骤(2)制备的CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S。
上述步骤(1)中CE7Q的制备方法,具体包括以下步骤:
1)将N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖CS-N3加入到二甲亚砜中,使CS-N3的终浓度为10 mg/mL,搅拌至完全溶解;然后加入Er、Q-amine和Cy7;
2)将步骤1)的体系置于氮气气氛中,往烧瓶中依次滴加五水硫酸铜的水溶液和维生素C钠的水溶液;用锡箔纸将烧瓶包住,室温搅拌三天;
3)取出步骤2)的反应液,放入8-14KDa的透析袋透析三天,取出透析液进行冷冻干燥,得到CE7Q。
N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖CS-N3,是通过“点击化学”反应将季胺盐修饰到壳聚糖反应得到的,具体见参考文献:Gao Y, Zhang Z, Chen L, Gu W, Li Y.Synthesis of 6-N,N,N-trimethyltriazole chitosan via "click chemistry" andevaluation for gene delivery. Biomacromolecules. 2009, 10(8):2175-82.。
上述步骤2)中CS-N3与Q-amine,Er,Cy7的质量比为:5∶4:1:1;
上述步骤3)中五水硫酸铜和维生素C钠的水溶液浓度分别为:20 mg/mL和 15 mg/mL,用量分别为 200 μL。
纳米复合物CE7Q/CQ/S在制备抗肿瘤药物中的应用。
纳米复合物CE7Q/CQ/S在输送基因药物中的应用。
纳米复合物CE7Q/CQ/S在制备近红外荧光成像剂中的应用。
其中,分子靶向药物厄洛替尼能特异性识别EGFR突变型细胞,光热剂Cy7能用于近红外荧光成像,基因药物SV能够抑制抗凋亡蛋白的表达。
本发明的作用原理是:
第一,壳聚糖作为带有正电荷的天然多糖,能够共输送化疗药物和基因药物并实现药物的控释,同时它具有较好的生物相容性,临床应用潜力大。
第二,利用分子靶向药物厄洛替尼识别不同分子分型的肺癌细胞,同时提高厄洛替尼的水溶性和治疗效果;
第三,Cy7不仅能用于近红外成像,还能用于光热治疗,促进药物释放的同时通过升高的温度杀死肿瘤细胞;
第四,通过基因沉默下调SV的表达,诱导肿瘤细胞凋亡,克服细胞对Er的耐药;
本发明的有益效果是:
第一,本发明以壳聚糖作为输送载体,通过控制纳米复合物的粒径实现被动靶向,将药物输送到肿瘤部位;
第二,本发明研发的纳米复合物具有较好的光热效果、激光和pH双重响应的释放特性和压缩基因并成功表达的能力;
第三.通过厄洛替尼的靶向作用和荧光分子Cy7的成像功能,识别不同分子分型的肺癌细胞;
第四,联合基因治疗和光热治疗协同逆转厄洛替尼的耐药问题,显著抑制肿瘤细胞的增殖,实现肺癌的高效治疗。
附图说明
图1为实施例1中 Er,Cy7,Q-amine,CS-N3和CE7Q的红外图谱。
图2为实施例1的CE7Q/CQ/S,实施例4的CEQ/CQ/S,实施例5的C7Q/CQ/S和实施例6的CQ/S的粒径分布。
图3为实施例7中CE7Q/CQ/S的释放特性图。
图4为实施例8中CE7Q/CQ/G的转染情况图。
图5为不同纳米复合物对三种不同分型肺癌细胞的体外毒性实验。
图6为实施例12中CE7Q/CQ/S在体内的光热效果。
具体实施方式
下面结合具体实施例,对本发明进行进一步说明,有助于本领域的普通技术人员进一步理解本发明,但不以任何形式限制本发明。
实施例1
将20 mg N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖CS-N3加入到2 mL二甲亚砜中,搅拌至完全溶解,再在烧瓶中加入4 mg Er、16 mg Q-amine和4 mg 的Cy7。上述体系置于氮气气氛中,用1 mL注射器往烧瓶中依次滴加200 μL五水硫酸铜的水溶液(20 mg/mL)和200 μL维生素C钠的水溶液(15 mg/mL)。用锡箔纸将烧瓶包住,室温搅拌三天。取出反应液,放入透析袋(8000~14000 Da)透析三天。取出透析液进行冷冻干燥,得到CE7Q。用ddH2O将CE7Q和CQ分别配成460 μg/mL和200 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CE7Q/CQ溶液,在涡旋状态下用移液枪吸取100 μL SV(80 μg/mL)溶液慢慢滴加到CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S。
本实施例制备所得CE7Q的红外图谱分析,见图1。图1表明Cs-N3在2100 cm-1处有很强的叠氮基团的吸收峰,而CE7Q中此处吸收峰明显减弱,这表明Er、Cy7和季铵盐已成功通过“点击化学”反应连接到壳聚糖链上。
本发明制备所得纳米复合物CE7Q/CQ检测其粒径大小,结果见图2所示,CE7Q/CQ/S的粒径为323.2±0.4 nm。
实施例2
用ddH2O将实施例1中的CE7Q和CQ分别配成100 μg/mL和200 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CE7Q/CQ溶液,在涡旋状态下用移液枪吸取100 μL SV(10 μg/mL)溶液慢慢滴加到CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S。
实施例3
用ddH2O将实施例1中的CE7Q和CQ分别配成1000 μg/mL和200 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CE7Q/CQ溶液,在涡旋状态下用移液枪吸取100μL SV(300 μg/mL)溶液慢慢滴加到CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S。
实施例4
将20 mg N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖加入到2 mL二甲亚砜中,搅拌至完全溶解,再在烧瓶中加入4 mg Er、16 mg Q-amine。上述体系置于氮气气氛中,用1 mL注射器往烧瓶中依次滴加200 μL五水硫酸铜的水溶液(20 mg/mL)和200μL维生素C钠的水溶液(15 mg/mL)。用锡箔纸将烧瓶包住,室温搅拌三天。取出反应液,放入透析袋(8000~14000Da)透析三天。取出透析液进行冷冻干燥,得到CEQ。用ddH2O将CEQ和CQ分别配成460 μg/mL和200 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CEQ/CQ溶液在涡旋状态下用移液枪吸取100 μL SV(80 μg/mL)溶液慢慢滴加到CEQ/CQ溶液中,室温孵育半小时,得到纳米复合物CEQ/CQ/S。检测其粒径大小,结果如图2所示,CEQ/CQ/S的粒径309.6±5.2 nm。
实施例5
将20 mg N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖加入到2 mL二甲亚砜中,搅拌至完全溶解,再在烧瓶中加入16 mg Q-amine和4 mg 的Cy7。上述体系置于氮气气氛中,用1 mL注射器往烧瓶中依次滴加200 μL五水硫酸铜的水溶液(20 mg/mL)和200 μL维生素C钠的水溶液(15 mg/mL)。用锡箔纸将烧瓶包住,室温搅拌三天。取出反应液,放入透析袋(8000~14000 Da)透析三天。取出透析液进行冷冻干燥,得到C7Q。用ddH2O将C7Q和CQ分别配成460μg/mL和200 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到C7Q/CQ溶液,在涡旋状态下用移液枪吸取SV(80 μg/mL)溶液100 μL慢慢滴加到C7Q/CQ溶液中,室温孵育半小时,得到纳米复合物C7Q/CQ/S。检测其粒径大小,结果如图2所示,C7Q/CQ/S的粒径413.6±0.7 nm。
实施例6
将CQ溶于ddH2O配制成浓度为100 μg/mL的溶液,在涡旋状态下用移液枪吸取100μLSV(80 μg/mL)溶液慢慢滴加到100 μL CQ溶液中,室温孵育半小时,得到纳米复合物CQ/S。检测其粒径大小,结果如图2所示,CQ/S的粒径267.9±2.2 nm。
实施例7
吸取2 mL的CE7Q/CQ/S溶液放入截留分子量为8000~14000 Da的透析袋中,再将透析袋放入20 mL含20wt%甲醇和1.25 mg/mL溶菌酶的PBS中。37℃搅拌条件下,在设定的时间取1 mL用于检测,同时补加1 mL的上述缓冲液。对于808 nm激光照射组,溶液在2 h和6 h时暴露于808 nm激光下照射5 min。CE7Q/CQ/S的释放特性曲线如图3所示,pH值为5.4时,48h Er的累积释放量为51.7%,808 nm NIR激光照射组中Er的累积释放量达到64.9%。在2 h和6 h,808 nm NIR激光照射能触发CE7Q/CQ/S中Er的快速释放,所以Er释放能被NIR激光控制。这是因为Cy7引起的溶液温度的升高促进了壳聚糖的离解。CE7Q/CQ/S在pH值为7.4的条件下释放量较少,在48 h时厄洛替尼Er的累积释放量为30%,这是因为溶菌酶在碱性环境中活性较低。在pH7.4条件下,Er在有激光照射时的累积释放量高于没有激光照射的累积释放量。
实施例8
用ddH2O将CE7Q和CQ分别配成920 μg/mL和400 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CE7Q/CQ溶液,在涡旋状态下用移液枪吸取100 μL GFP shRNA(160 μg/mL)溶液慢慢滴加到100 μL CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/G,用于后续的细胞转染实验。
实施例9
用ddH2O将CE7Q和CQ分别配成920 μg/mL和400 μg/mL的溶液,然后各吸取50 μL放入离心管中,混合均匀后得到CE7Q/CQ溶液,在涡旋状态下用移液枪吸取100 μL SV(160 μg/mL)溶液慢慢滴加到100 μL CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S,用于后续的细胞毒性实验。
实施例10
本实验选择A549细胞(EGFR野生型,Er原发性耐药型)、PC-9(EGFR突变型,Er敏感型)和H1975细胞(EGFR的L858R和T790M突变,Er突变耐药型)为测试细胞系。
细胞培养方法:将A549,PC-9和H1975细胞的保种管从液氮罐中取出,迅速将保种管底部放入37℃水浴锅中,不断摇动,待冻存液融化后,在1000转/min的条件下离心5 min;弃去冻存液,用1 mL培养液将细胞沉淀吹打均匀后转移至培养瓶中,再向培养瓶中补加3mL 培养基,将培养瓶置于37℃,5% CO2培养箱中培养。
细胞转染实验:按照每孔4×104个细胞将对数生长期的A549、PC9和H1975细胞接种在24孔板中,待细胞贴壁后,用Lipofectamine® 2000运载GFP pDNA得到的L/G转染三种肺癌细胞4小时后,生理盐水洗2遍,加入新鲜的培养基继续培养24 h后,得到表达GFP蛋白的三种肺癌细胞。用实施例6制备的CE7Q/CQ/G孵育表达GFP蛋白的三种肺癌细胞2 h后,生理盐水洗2遍,加入新鲜的培养基继续培养24 h,在倒置荧光显微镜下观察GFP的表达情况。
结果如图4所示,相比于对照组,三种细胞CE7Q/CQ/G实验组GFP的荧光均变少,证明CE7Q/CQ具有运载基因药物进入细胞并顺利表达的能力。其中,CE7Q/CQ/G对PC9细胞的转染效率高,达到84.9±1.12%;对A549和H1975细胞的转染效率稍差,分别为78.53±3.13%和69.3±1.85%,这是因为耐药细胞对药物的摄取量略有下降。
实施例11
细胞毒性实验:按照每孔8000个细胞将生长状态良好的肺癌细胞接种在96孔板中,放入培养箱培养24 h,吸弃旧的培养基,加入实施例7制备的CE7Q/CQ/S纳米复合物(Er的浓度为8 μg/mL,Cy7的浓度为6 μg/mL,SV的浓度为10 μg/mL),对照组加入与CE7Q/CQ/S纳米复合物含相同SV基因浓度的CQ/S、含相同Er浓度的CEQ/CQ/S以及含相同Cy7浓度的C7Q/CQ/S,照射组给予808 nm NIR激光照射(功率:1.0 W/cm2,时间:5 min)。培养2 h,然后换新鲜的含10%血清的无含药RPMI-1640培养基继续培养24 h。100 μL的MTT溶液孵育4 h。用移液枪吸走MTT,加入150 μL DMSO溶液,溶解甲瓒。然后将96孔板平放在摇床上以50 rpm的速度振荡10 min并保持37℃恒温。最后在酶联免疫检测仪中检测570 nm波长处的吸收值。并按下式计算细胞的存活率。
存活率(%)=(实验组吸收值-溶剂对照组吸收值)/(空白组吸收值-溶剂对照组吸收值)。
结果如图5所示,CQ/S,CEQ/CQ/S,C7Q/CQ/S和CE7Q/CQ/S对三种细胞的增殖均具有抑制作用。在同一SV浓度下,CQ/S对PC9细胞的毒性较大,对A549和H1975两种细胞毒性较小,这是由于CQ/S对耐药细胞的转染效率稍低。当Er含量相同时,相比与CEQ/CQ/S,CE7Q/CQ/S实验组细胞存活率更低,这因为Cy7能定位到溶酶体,提高了Er在细胞内的有效浓度,且Cy7本身对三种细胞的增殖具有抑制作用,三种细胞中CE7Q/CQ/S对PC9细胞治疗效果最好。相同Cy7浓度下,CE7Q/CQ/S对细胞增殖的抑制作用比C7Q/CQ/S更强,是因为Er与基因药物的联合,提高了细胞对Er的敏感性。在三种细胞中,CE7Q/CQ/S +NIR对细胞的毒性比CE7Q/CQ/S强,这是因为SV、Er以及光热治疗的协同作用,有效克服了Er的耐药性。
实施例12
H1975裸鼠皮下肿瘤模型的构建:取生长状态良好的H1975细胞用胰酶消化后制成细胞悬浮液(3×107个/100 μL),向裸鼠皮下注射细胞悬液100 μL,置于清洁无菌环境下饲养。
待H1975裸鼠的肿瘤体积达到100 mm3后,每只裸鼠瘤内注射生理盐水、Cy7、C7Q/CQ/S和CE7Q/CQ/S。每组给药4 h后,对肿瘤部位进行近红外激光照射(808 nm,1.0 W/cm2),照射时间5 min,并用近红外热像仪进行拍照记录。结果如图6所示,给药组老鼠的肿瘤的温度均随照射时间的增加而增加,照射5 min后,Cy7组裸鼠肿瘤部位的温度达到55.6℃,C7Q/CQ/S组裸鼠肿瘤部位的温度达到60.8℃,而CE7Q/CQ/S组达到59.1℃,说明纳米复合物可实现光热治疗,并能提高Cy7的光热转换效果。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (8)
1.一种兼具分子靶向/基因/光热治疗的纳米复合物,其特征在于:该纳米复合物是通过在壳聚糖骨架上偶联分子靶向药物厄洛替尼Er、荧光分子Cy7和炔丙基季铵溴盐Q-amine得到CE7Q,CE7Q与季铵盐修饰的壳聚糖CQ混合后,共负载基因药物Survivin shRNA的表达质粒SV,得到的纳米复合物CE7Q/CQ/S;
CE7Q、CQ和SV的质量比为0.5:1:0.1~5:1:3。
2.一种制备如权利要求1 所述的兼具分子靶向/基因/光热治疗的纳米复合物的方法,其特征在于:包括以下步骤:
(1)用ddH2O分别将CE7Q 、CQ和SV配置成溶液;
(2)然后按质量比将CE7Q溶液 、CQ溶液混合均匀后得到CE7Q/CQ溶液;
(3)在涡旋状态下按质量比将SV溶液慢慢滴加到步骤(2)制备的CE7Q/CQ溶液中,室温孵育半小时,得到纳米复合物CE7Q/CQ/S。
3.根据权利要求2所述的方法,其特征在于:所述步骤(1)中CE7Q的制备方法,包括以下具体步骤:
1)将N-4-叠氮-邻苯二甲酰亚胺基-壳聚糖CS-N3加入到二甲亚砜中,使CS-N3的终浓度为10 mg/mL,搅拌至完全溶解;然后加入Er、Q-amine和Cy7;
2)将步骤1)的体系置于氮气气氛中,往烧瓶中依次滴加五水硫酸铜的水溶液和维生素C钠的水溶液;用锡箔纸将烧瓶包住,室温搅拌三天;
3)取出步骤3)的反应液,放入8-14KDa的透析袋透析三天,取出透析液进行冷冻干燥,得到CE7Q。
4.根据权利要求3所述的方法,其特征在于:所述步骤1)中CS-N3与Q-amine,Er,Cy7的质量比为:5∶4:1:1。
5.根据权利要求3所述的方法,其特征在于:上述步骤2)中五水硫酸铜和维生素C钠的水溶液浓度分别为:20 mg/mL和15 mg/mL,用量分别为 200 μL。
6.根据权利要求1所述的纳米复合物在制备抗肿瘤药物中的应用。
7.根据权利要求1所述的纳米复合物在制备输送基因药物中的应用。
8.根据权利要求1所述的纳米复合物在制备近红外荧光成像剂中的应用。
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