CN117247906A - 一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料 - Google Patents
一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料 Download PDFInfo
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Abstract
本发明公开了一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,重组细胞膜上高表达目标膜受体,其N端融合表达Halo‑tag蛋白标签;重组细胞膜通过Halo‑tag蛋白标签共价结合、反向包覆在氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒上。本发明制备高密度FGFR4细胞膜药物筛选材料,增强目标膜受体FGFR4对活性化合物的特异性筛选能力,提高筛选方法的选择性和灵敏度。
Description
技术领域
本发明属于细胞膜药物筛选技术领域,涉及一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料。
背景技术
中药是中华民族的文化瑰宝,它凝聚了几千年来中国人民防病治病和养生保健的智慧。中药有效部位或成分是中药发挥药理作用的物质基础,它既揭示了中药在体内的作用规律,也增强了中药的临床药理作用,并且对于新药以及中药新产品的研发具有重要意义。但是中药目前存在着产地来源多样、炮制方法繁琐、组成成分复杂且作用机制不明确等问题。并且由于中药成分复杂,目前很多中药的有效成分还没有完全研究清楚,因此在实际研究中往往会选择已知并且易得的标志性化学成分,但是这些成分并不真正是中药的活性成分。因此,如何辨识和筛选出能够代表中药治疗效果的活性成分成为中药现代化研究的关键问题之一。
近年来,以生物材料为靶标筛选中药活性成分的研究方法成为热点,这种方法具有针对性强、灵敏度高的优点,还能辅助阐明药物的作用机制。其中,由于细胞膜具有能够复制生命系统单元整体表面特性的能力,因此以细胞膜为筛选工具的研究越来越受到关注。研究表明,至少60%的药物是与其特定的细胞膜受体相互作用从而发挥药效的,因此细胞膜受体是药物发现的主要靶标。细胞膜仿生材料显示出许多理想的特征,例如特异性靶向能力,膜蛋白的天然结构的保留和单层膜覆盖。随着细胞膜仿生材料研究工作的不断深入与扩展,将其应用于中药复杂体系的药物筛选体现出灵敏和快速等特点。
色谱方法-细胞膜色谱法(cell membrane chromatography,CMC),将含靶标受体的活性细胞膜作为固定相,用液相色谱法研究药物与固定相上细胞膜受体的相互作用。该方法可以最大限度地保持细胞膜的整体性以及膜受体的立体结构和生物活性。中药复杂体系可不经分离步骤,直接在细胞膜色谱上实现活性筛选过程,这种基于多组分-多靶标相互作用的方法非常适用于中药复杂体系活性成分筛选研究。近年来,利用CMC法,对大量中药进行了活性成分的筛选。当固定相无机载体采用磁性纳米材料时,用细胞膜涂层磁性纳米材料所建立的细胞膜磁性微球固相萃取方法能够为药物筛选材料提供具有良好的磁响应性和特异性亲和能力,使细胞膜磁性材料与中药总提物快速分离。
细胞膜药物筛选方法以细胞膜涂层无机载体为技术路线,以活性筛选为导向,以中药复杂体系为研究目标,以分离目标组分为原则,实现了中药物质基础分析,具有特异性高,操作简便且作用机制明确的优点。然而细胞膜包裹微纳米材料的药物筛选方法都是基于生物材料的分析方法,其核心部件-细胞膜的活性决定了整个分析方法的筛选效能和应用价值。但是由于细胞膜表面表达多种膜受体,在制备过程中载体材料表面涂覆的细胞膜是非选择性的,导致最终制备的细胞膜仿生材料表面存在着多种能和不同药物结合的膜受体,降低了目标膜受体的纯度,影响目标膜受体对活性化合物的特异性筛选能力,削弱筛选方法的选择性。
近年来,基于基因工程的细胞膜仿生纳米载体的仿生设计策略发展迅速。通过基因修饰可以赋予细胞膜额外的功能配体,使得细胞膜仿生材料可以满足多功能和多任务的复杂生物系统的应用要求。亲和标签融合技术是利用基因工程技术将改造优化的亲和标签与目标蛋白融合表达,从而对目标蛋白进行荧光标记实时成像或者纯化的技术,它具有结合特异性强及通用性强的优点。其中,Halo-tag是由细菌脱卤素酶(haloalkanedehalogenase,halo)改造的蛋白标签,可以特异性地与连接有不同功能基团的氯代烷烃配基共价结合。通过Halo-tag蛋白标签与目的蛋白构建融合表达载体,能够保证遗传标记的特异性,并通过改变Halo-tag配基实现不同的研究目的。
发明内容
本发明解决的技术问题在于提供一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,增强目标膜受体FGFR4对活性化合物的特异性筛选能力,提高筛选方法的选择性和灵敏度。
本发明是通过以下技术方案来实现:
一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,重组细胞膜上高表达目标膜受体,其N端融合表达Halo-tag蛋白标签;
重组细胞膜通过Halo-tag蛋白标签共价结合、反向包覆在氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒上。
所述重组细胞膜上的目标膜受体为FGFR4受体;
重组细胞膜是反向包覆在氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒上,充分暴露受体的酪氨酸激酶区域。
所述的Fe3O4@SiO2磁性纳米颗粒上还连接有链霉亲和素。
所述的氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒通过以下途径制得:
1)通过法制备Fe3O4@SiO2磁性纳米粒子;
2)将Fe3O4@SiO2-NH2纳米颗粒羧基化得到Fe3O4@SiO2-COOH纳米颗粒;
3)将Fe3O4@SiO2-COOH纳米颗粒活化后与链霉亲和素共同孵育后,分离得到Fe3O4@SiO2-SA磁性纳米颗粒;
4)将Fe3O4@SiO2-SA磁性纳米颗粒与包含HaloTag配基的氯代烷烃修饰剂充分混合震荡,得到氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒。
所述的重组细胞膜来自于慢病毒介导的含Halo-tag标签的FGFR4重组质粒转染HaCat细胞;
所述的含Halo-tag标签的FGFR4重组质粒是将含有Halo-tag标签的编码区插入FGFRR4的N端。
所述的重组质粒为重组质粒LV-EFS>Halo tag/hFGFR4/3xEAAAK/EGFP-PGK>Puro。
所述的高密度细胞膜受体包覆的细胞膜磁性纳米材料在筛选抗肿瘤药物中的应用。
所述是从包括虎杖在内的中药中筛选抗肿瘤的活性化合物。
与现有技术相比,本发明具有以下有益的技术效果:
本发明提供的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,通过对目标膜受体亲和标签融合技术修饰,提高了重组目标膜受体的产量;利用细胞膜受体N-端融合的Halo-tag蛋白标签,将含有目标膜受体的细胞膜片段特异性共价结合到氯代烷烃修饰的载体材料表面,保证了细胞膜药物筛选材料表面FGFR4的高密度;保证了细胞膜药物筛选材料上受体结合位点的全暴露,提高了方法对目标物质的选择性。
附图说明
图1-1为重组质粒LV-EFS>Halo tag/hFGFR4/3xEAAAK/EGFP-PGK>Puro的元件排列示意图;
图1-2为重组细胞FGFR4表面高密度验证结果;
其中,A为FGFR4在不同细胞组中的表达。(a)和(b)分别为Western blot检测FGFR4表达(1:高HFE Hacat细胞膜组;2:NC-Hacat细胞膜组)。(c)为FGFR4 mRNA的相对表达量(1:高HFE Hacat细胞组;2:NC-Hacat细胞组)。
B为共聚焦显微镜图像。(SA-APC标记的红色荧光表示HFE细胞膜的存在,DAPI标记的蓝色荧光表示细胞核的存在(标尺=15μm)。
C为Western blot检测结果,(a)和(b)Western blot检测FGFR4表达(1:HDFGFR4/CMMNPs;2:FGFR4/CMMNPs)。
D为共聚焦显微镜图像。(SA-APC标记的红色荧光表示HFE细胞膜的存在,FITC标记的绿色荧光表示MNPs的存在(标尺=15μm)。
统计学差异采用Student’s t检验,差异有统计学意义:*p<0.05anddramatically significance:***p<0.01vs.阴性对照组。
图2为HDFGFR4/CMMNPs的物理化学特征;
其中,A为免疫金标示细胞外或细胞内CD47结构域的HDFGFR4/CMMNPs、FGFR4/CMMNPs和裸MNPs的透射电镜图像。
B为HDFGFR4/CMMNPs和裸MNPs的SEM图像。
C为HDFGFR4/CMMNPs共聚焦显微镜图像:DiI标记的红色荧光(a、C和e)代表细胞膜的存在,FITC标记的绿色荧光(b、d和f)代表MNPs和(标度条=2.5μm)的存在。
D为统计结果;(a)高HFE Hacat细胞膜衍生囊泡的尺寸和zeta电位结果,(b)Fe3O4-COOH纳米颗粒,(c)HDFGFR4/CMMNPs,(D)FGFR4/CMMNPs。
图3为裸MNPs(a)和HDFGFR4/CMMNPs(b)的VSM曲线。
图4为Fe3O4(a)、裸MNPs(b)和(c)HDFGFR4/CMMNPs的XRD谱图。
图5为HDFGFR4/CMMNPs在的吸附能力表征。
其中,A为HDFGFR4/CMMNPs和FGFR4/CMMNPs的静态吸附。
B、C分别为:HDFGFR4/CMMNPs和FGFR4/CMMNPs的Langmuir等温线和Freundlich等温线结果。
D为五种化合物在HDFGFR4/CMMNPs和FGFR4/CMMNPs上的释放百分比。
E为HDFGFR4/CMMNPs和FGFR4/CMMNPs的动力学吸附结果。
F为洗脱液选择:(a)水,(b)50mmol L-1NH4CH3COOH,(c)50mmol L-1PBS(d)DMSO-50mmol L-1PBS(1:99,v/v),(e)DMSO-50mmol L-1PBS(5:95,v/v),(F)DMSO-50mmol L-1PBS(15:85,v/v)。G:HDFGFR4/CMMNPs使用量。
H为洗脱时间。
图6筛选中总萃取液、上样液、洗涤液和洗脱液的色谱图;
其中,A为HDFGFR4/CMMNPs预处理虎杖提取物的色谱图:(A)初始提取物溶液,(b)上样后溶液,(c)洗涤后溶液,(d)洗脱后溶液。
B为淋洗液中峰1、峰2和峰3的TOFMS和化学结构。
C为CCK-8检测HepG2细胞活力的结果(n=3)。
D、E分别为0、12、24、48h的划痕实验(n=3)。
C、E采用Student’s t检验,差异有统计学意义:*p<0.05and dramaticallysignificance:***p<0.01vs.阴性对照组。
图7.为CCK-8检测HepG2细胞的细胞活力(n=3),差异采用Student’s t检验,差异有统计学意义:*p<0.05and dramatically significance:***p<0.01vs.阴性对照组。
图8为0、12、24和48划痕实验(n=3);
图9为定量分析0、12、24、48h时划痕试验结果(n=3),差异采用Student’s t检验,差异有统计学意义:*p<0.05and dramatically significance:***p<0.01vs.阴性对照组。
图10为赤松素、BLU9931的细胞凋亡分析结果(n=3)。
图11为虎杖苷、大黄素的细胞凋亡分析结果(n=3)。
具体实施方式
下面结合实施例对本发明做进一步详细描述,所述是对本发明的解释而不是限定。
本发明利用目标膜受体N-端融合的Halo-tag蛋白标签与氯代烷烃修饰的载体材料之间的共价结合作用,将位于细胞膜上高表达成纤维细胞生长因子受体4(fibroblastgrowth factors receptor 4,FGFR4)的Halo-tag位点特异性地固定在载体材料表面,制备高选择性细胞膜药物筛选材料。
FGFR4属于受体型蛋白酪氨酸激酶家族,临床研究发现很多肿瘤的发生都伴随着肿瘤组织的FGFR4过表达和激活。因而FGFR4可以被用来作为筛选抗肿瘤药物的靶标。通过将目标细胞膜受体FGFR4的基因工程修饰,再将位于细胞膜上高表达FGFR4受体的Halo-tag位点特异性地固定在载体材料表面,制备高纯度FGFR4细胞膜药物筛选材料用于中药中活性化合物的快速筛选。
1、FGFR4高表达细胞膜药物筛选磁性材料的制备
(1)氯代烷烃功能化磁性载体的制备
①采用法制备Fe3O4@SiO2磁性纳米粒子:
3.60g FeCl3·H2O和0.72g柠檬酸三钠超声溶解在100mL乙二醇中,高速搅拌,加入4.80g NaAc,50℃剧烈搅拌30min后,将混合物密封在聚四氟乙烯衬里的不锈钢高压釜中,200℃下反应10h后,将所得产物分别用甲醇和水洗涤三次可得Fe3O4@SiO2磁性纳米粒子。
取0.1g超声分散后的Fe3O4磁性纳米颗粒加入80mL乙醇和12mL超纯水的混合溶剂中,超声分散后在机械搅拌下加入4mL,25%氨水溶液,将0.4mL TEOS逐滴加入混合体系中,40℃搅拌8h,用外部磁场分离制得的Fe3O4@SiO2纳米颗粒,将所得产物分别用甲醇和水洗涤三次,洗至溶液呈中性。
②Fe3O4@SiO2-COOH磁性纳米粒子的制备:
首先制备Fe3O4@SiO2-NH2纳米颗粒。将0.4g Fe3O4@SiO2纳米颗粒分散在50mL无水甲苯中,超声处理30min后加入4mL 1,3-氨基丙基三乙氧基硅烷(APTES),N2保护下120℃搅拌24h,充分洗涤后冷冻干燥可得100mg Fe3O4@SiO2-NH2纳米颗粒。
将制备得到的Fe3O4@SiO2-NH2转移至50mL两颈圆底烧瓶中,加入10mL甲苯,40mg丁二酸酐,在氮气保护下,加热至80℃,待丁二酸酐全部溶解后,继续磁力搅拌12h。将得到的产物分别用甲苯和无水乙醇清洗3次,最后用丙酮清洗2次,吹干丙酮后,55℃真空干燥12h,获得羧基化(Fe3O4@SiO2-COOH)磁性载体材料。
③Fe3O4@SiO2-SA磁性纳米粒子的制备:
将10mg Fe3O4@SiO2-COOH纳米颗粒用0.1mol/L 2-(N-吗啉)乙磺酸(MES)缓冲液(pH=5.2)洗涤3次,然后在0.1mol/L EDC MES缓冲液在室温下振荡激活30min。
为了避免活化后的羧酸官能团水解,快速用新鲜MES缓冲液淋洗几次活化后的Fe3O4@SiO2-COOH纳米颗粒,将活化后的Fe3O4@SiO2-COOH纳米颗粒与450μg链霉亲和素(Streptavidin,SA)共同在室温下孵育24h。孵育结束后,用甘氨酸溶液(1.0mol/L,pH=8.0)中和过量的SA,接着用PBS磷酸缓冲盐淋洗制备的纳米颗粒。最后,将所得产物分别用甲醇和水洗涤三次,洗至溶液呈中性,冷冻干燥后即可成功制备出Fe3O4@SiO2-SA磁性纳米颗粒。
④氯代烷烃功能化磁性载体的制备
将5mg Fe3O4@SiO2-SA磁性纳米颗粒,用300μL 1×TBS溶液充分洗涤后,投入氯代烷烃修饰剂,比如1μmol/LPEG-Biotin Ligand(promega),充分混合后,室温下震荡混合30min,反应结束后使用300μL 1×TBS溶液充分洗涤五次,即可得到氯代烷烃功能化磁性载体(Chloroalkane-functionalized magnetic nanoparticles,CMNPs);
HaloTag蛋白标签能够和氯代烷烃修饰的载体特异性亲和,所以用氯代烷烃修饰磁性纳米粒子,为后续选组性吸附HaloTag标记的FGFR4受体做准备。
(2)慢病毒介导的目的基因转染HaCat细胞研究
①含Halo-tag标签的FGFR4慢病毒表达载体的构建:
将含有Halo-tag标签的编码区插入FGFRR4的N端:
具体的,将质粒载体(LV-PGK>Puro)分别在37℃水浴中进行AscI和BamHI双酶切反应,1%琼脂糖凝胶电泳回收AscI+BamHI酶切后的质粒大片段。回收大片段与人FGFR4(NCBI参考序列:NM_213647.3)基因酶切重组,重组反应在50℃反应15min;构建得到重组质粒LV-EFS>Halo tag/hFGFR4/3xEAAAK/EGFP-PGK>Puro(其元件排列如图1-1所示);
重组质粒挑取3个单菌落接种于Ampicillin抗性的培养液中,培养过夜后,用小量质粒抽提试剂盒抽提质粒,质粒用ApalI+EcoRI进行酶切鉴定,挑取阳性克隆进行测序验证,将鉴定正确的重组质粒用于下一步细胞转染。
②含Halo-tag标签的FGFR4的HaCat细胞转染:
将重组质粒进行慢病毒包装,在转染前2天传代准备细胞:用0.25%胰蛋白酶消化293T细胞,10%血清的DMEM培养基培养48h,当细胞密度增加至80%以上时即可用于转染。
在转染前用5%血清不含抗生素的DMEM培养基换液。将重组质粒导入293T细胞,产生高滴度含Halo-tag-FGFR4基因的慢病毒,完成病毒包装。
取培养后的Hacat细胞,用计数板调整细胞密度并接种于六孔板,分别加入含Halo-tag-FGFR4基因的慢病毒75μL,5%CO2、37℃培养24h。分别在转染24、48、72h进行荧光显微拍照(100×)。为了获得稳定表达Halo-tag-FGFR4的Hacat细胞系,转染48h后将细胞转移至10cm培养皿进行培养,更换为含嘌呤霉素(8mg·L-1)的新鲜培养基,重复3次,即可得到稳定转染Halo-tag-FGFR4的Hacat细胞。
③含Halo-tag标签的高表达FGFR4 HaCat细胞培养;
为了得到阳性表达的HaCat细胞,选择生长状态良好的转染后的HaCat细胞,胰酶消化后,用PBS缓冲液清洗两遍,取无菌离心管,加入20%进口血清培养基5mL,收集分选的细胞。同时用含5%双抗的PBS缓冲液高速清洗分选管路15min,QC beads调节收集管的位置后用流式细胞仪上机分选并用FlowJo 7.6.1软件进行数据分析,将收集管中的细胞离心5min后接种于24孔板培养。培养7d后,待细胞生长融合至80%状态时,将细胞转移至培养皿培养。分选后膜外区含Halo-tag标签的高表达FGFR4HaCat细胞在10%胎牛血清,100U·mL-1链霉素,300mg·L-1遗传霉素和100U·mL-1青霉素的DMEM培养基中培养,在37℃,含5% CO2的潮湿气氛中生长。
④实时定量PCR检测mRNA:
实验采用转染LV-PGK>Puro的HaCat细胞作为阴性对照,对制备的高表达FGFR4的HaCat细胞膜进行实时荧光定量PCR检测。首先利用Trizol法提取细胞中的RNA,并将其逆转录成cDNA,并进行稀释,在50℃20min,95℃10min,95℃30s,60℃2030s的条件下进行40个循环。并对所的结果进行分析,判断目的基因表达情况。
(3)含Halo-tag标签的高表达FGFR4 HaCat细胞膜制备
①含Halo-tag标签的高表达FGFR4 HaCat细胞膜制备:
收集来自指数期生长的膜外区含Halo-tag标签的高表达FGFR4 HaCat细胞,并用生理盐水(pH=7.4)洗涤三次以准备制备膜外区含Halo-tag标签的FGFR4细胞膜制剂。
当细胞生长状态贴近80%时,进行计数,取细胞数目不低于107的细胞用0.25%胰蛋白酶消化得到单细胞混悬液。收集细胞,于4℃条件下1000g离心10min去除DMEM细胞培养液,用生理盐水重新混悬后,重复清洗三次。取细胞沉淀加入5mL 50mmol·L-1Tris–HCl溶液(pH=7.4)重新混悬,4℃下冰浴超声破碎细胞30min,将悬液于4℃条件下1000g离心10min,取上清液于4℃下12000g离心20min,即可得细胞膜沉淀物。将细胞膜沉淀用生理盐水重新混悬,再次于4℃下12000g离心20min,最终得到高表达膜外区含Halo-tag标签的FGFR4细胞膜制剂。
②表征考察:
通过MIR、DLS、液质联用(LC-MS)、Western Blot等方法对高表达膜外区含Halo-tag标签的FGFR4细胞膜制剂的粒径和电势进行考察,确保制备出性能优异的细胞膜制剂。
(4)位点特异性细胞膜涂层
由于细胞膜表面的FGFR4上膜外区的Halo-tag标签,可以利用Halo-tag与载体材料表面的Halo-tag配基的特异性共价亲和作用,可以将具有高表达FGFR4受体的细胞膜特异性固定在载体材料表面,充分暴露受体的酪氨酸激酶区域(图2的A免疫金电镜实验就是证明细胞膜是反向包覆的,可以暴露懒氨酸激酶区域)。
将5mg氯代烷烃功能化磁性纳米粒子与1mL膜外区含Halo-tag标签的FGFR4细胞膜制剂冰浴超声震室温下震荡混合2-4h,混合结束后使用甘氨酸溶液(1.0mol/L,pH=8.0)中和过量的生物素,再次用预冷的PBS溶液充分洗涤目标膜受体的特异性固定的细胞膜包裹磁性纳米粒子。
利用载体材料表面的修饰氯代烷烃功能基团和细胞膜膜外区的Halo-tag标签之间的特异性结合能力,再在脂质体挤出器的聚碳酸酯多孔膜反复挤压作用下,把细胞膜涂覆在磁性纳米载体表面,得到FGFR4的特异性固定的细胞膜包裹磁性纳米粒子CMMNPs。
2、FGFR4高表达细胞膜药物筛选模型的建立
(1)细胞膜药物筛选磁性材料的目标膜受体纯度考察
通过Western Blot实验验证CMMNPs(CMNPS@CM)组以及对照组MNPs@CM中的FGFR4表达情况,计算各组蛋白条带相对值,分析融合蛋白表达情况。
具体地,收集材料,RIPA裂解液在冰上裂解60min后取上清液测定BCA蛋白总量。按照试剂盒分别配制浓缩胶和分离胶,每孔30μg总蛋白进行SDS-PAGE凝胶分离,然后将含有目的条带的凝胶转移至甲醇活化后的PVDF膜上。将PVDF膜在5%的脱脂奶粉的封闭液中封闭1h,然后震荡漂洗,按照说明书稀释一抗,加入FGFR4(1:1000)一抗和β-actin(1:1000)一抗,4℃下摇床孵育过夜。TBST溶液漂洗4次后,加入稀释后的HRP标记的羊抗鼠IgG二抗(1:10000),37℃摇床孵育2h后,TBST溶液漂洗4次,加入化学发光液ECL温浴后曝光和显影。用Quantity One分析软件进行分析,计算各组蛋白条带相对值,分析融合蛋白表达情况。
(2)细胞膜药物筛选磁性材料的性能
①细胞膜药物筛选材料的吸附性能考察:
通过考察CMMNPs的静态和动态吸附性能,可以评价所制备的药物筛选材料的吸附平衡速率、吸附容量和吸附类型等特性。配制BLU9931(FGFR4的选择性抑制剂)的对照品溶液,通过静态吸附试验和动态吸附试考察CMMNPs和对照组对BLU9931的吸附容量和吸附数据等参数,并对所得数据用不同的吸附模型进行拟合,根据最适吸附模型进一步进行分析评估,判断吸附过程的特性。
②细胞膜药物筛选材料的特异性考察:
细胞膜药物筛选材料的特异性是评价CMMNPs的重要参数。通过对筛选材料特异性的考察可以评价材料的对不同化合物的亲和力。分别配置作用于不同受体包括FGFR4在内药物的对照品溶液,与CMMNPs细胞膜磁性药物筛选材料充分震荡混合并计算CMMNPs对每种对照品的吸附量,评价所制备材料对其阳性药物的特异性吸附能力。
(3)药物筛选方法的建立
①细胞膜药物筛选方法的建立:
本发明以BLU9931为目标分子,首先配制BLU9931以及阴性药物的对照品溶液,将成功制备的CMMNPs药物筛选材料投入到各对照品溶液中,按照萃取条件,分步骤进行吸附,通过外加磁场将药物筛选材料与液体基质分离,最终将所筛选出的活性化合物进行解吸,计算筛选效率,评价所建立方法的有效性和效率。
②方法学考察:
为了验证所建立方法的可靠性,对所建立方法的线性关系、定性/定量检测限、加标回收率、精密度、重现性和稳定性等方法学指标进行考察。
(4)虎杖等中药中活性化合物的筛选
将成功制备的CMMNPs药物筛选材料实际应用到虎杖等中药总提物的活性成分筛选工作中,按照前期确定的萃取条件,分步骤进行吸附,通过外加磁场将药物筛选材料与中药复杂基质分离,最终将所筛选出的活性化合物进行解吸,得到中药总提物中可与FGFR4发生特异性作用的活性成分。
对解析液中可与FGFR4发生特异性作用的活性成分进行分离和鉴定,首先对解析液利用HPLC-TOFMS进行分析确定活性成分,利用pre-HPLC对活性成分进行分离纯化得到单体化合物,并对所得的单体化合物进行质谱分析、对照品比对,验证化合物的结构。
(5)活性化合物的筛选
(1)活性化合物的分子生物学和细胞生物学作用机制
①细胞增殖抑制实验:
发明采用CCK-8法验证筛选的活性成分对高表达FGFR4的HaCat细胞的增殖抑制毒性。首先收集在对数期生长的细胞并将其接种于96孔板中,细胞接种浓度为7000个/孔并且于37℃培养箱中孵育24h。配制不同浓度梯度的活性化合物培养基溶液并将其加入细胞孔板中孵育48h,每孔加入20μL CCK8并且在37℃环境下孵育30min,用酶标仪在490nm下测定对应的吸光度。根据所得结果绘制得到细胞生长曲线,并算IC50值,根据结果判断活性成分对细胞的增殖抑制作用。
②划痕实验:
取对数增长期的高表达FGFR4的HaCat细胞接种于12孔板,细胞接种浓度为4×105个/孔,并且于37℃,5%CO2恒温培养箱中培养过夜。待细胞增长至80%融合状态时,用200μL的枪头在孔中划一道划痕,注意保持宽度一致,用PBS缓冲溶液淋洗后加入新鲜培养基,拍照,记录为0h。然后分别加入不同浓度的筛选出的活性化合物培养基溶液,对照组加入等量的无血清培养基。分别于24h,48h,72h时拍照记录,分析不同时间下各个划痕上的细胞距离,判断活性化合物对细胞迁移的影响。
③流式细胞术:
取对数增长期的高表达FGFR4的HaCat细胞接种于6孔板,细胞接种浓度为6×105个/孔。在37℃,5% CO2恒温培养箱中培养24h,然后加入不同浓度的活性化合物培养基溶液,将培养板于37℃,5% CO2恒温培养箱中孵育培养。48h后,PBS缓冲溶液冲洗3次,0.25%胰蛋白酶消化细胞,1000rpm,5min离心细胞悬液,用PBS缓冲溶液清洗3次,加入200μL结合缓冲液重悬细胞。5μL Annexin V-FITC溶液室温下避光孵育5min,再加入10μL 20μg/mL的PI染色液,室温避光孵育10min,加入300μL结合缓冲液,轻轻混合均匀后用流式细胞仪检测,分析筛选出的活性化合物对细胞生长状态的影响。
④分子对接实验
为了模拟预测目标组分与对应受体的作用位点与作用模式,并且预测筛选出的化合物与相应受体作用的亲和力大小,本实验通过分子对接实验,使用Sybyl-X 2.0的Surflex-Dock模块来对筛选出的活性组分的抗肿瘤潜在作用机制进行研究。络氨酸激酶FGFR4的X射线晶体结构从蛋白质数据库中检索获得,并且蛋白质结构中的抑制剂以及水分子被去除,同时添加氢原子,用Powell的方法来优化,然后使用Gasteiger-Hückel电荷和Tripos立场来最小化能量,最后将化合物对接到刚性受体蛋白中。
3、实验验证
3.1FGFR4表面高密度确证
为了研究Halo标记的FGFR4高表达(Halo-tagged FGFR4 expression,HFE)Hacat细胞的成功构建,我们进行了western blot分析和聚合酶链反应(polymerase chainreaction,qPCR)实验。如图1-2中A的(a)和(b)所示,与Hacat细胞膜(对照,没有质粒转染的原本的母细胞)组相比,高HFE Hacat细胞膜明显表现出FGFR4的表达。采用qPCR检测基因表达,结果如图1-2的(c)所示。显然,与Hacat细胞组相比,高HFE Hacat细胞组FGFR4表达上调521倍。结果表明,高HFE Hacat细胞成功构建。
除了定量western blot和qPCR检测外,还通过共聚焦显微镜分析进行了定性研究。为了进一步证明高HFE Hacat细胞的成功构建,我们使用异藻蓝蛋白共轭链亲和素(llophycocyanin conjugated streptavidin,SA-APC,激发/发射光谱:647nm/666nm)对生物素配体(promega,Madison,USA)预孵育的高HFE Hacat细胞进行染色,同时使用4′,6-二氨基-2-苯基吲哚(4',6-diamidino-2-phenylindole,DAPI)染色细胞核。
图1-2的B显示,SA-APC的红色荧光信号和dapi染色的细胞核的蓝色荧光信号非常清晰,可见高HFE的Hacat细胞。阴性对照(negative control,NC)-Hacat细胞则没有红色荧光信号,这表明HaloTag在细胞膜上成功实现了高表达。
然后利用Halo标记的FGFR4高表达Hacat细胞膜制备HDFGFR4/CMMNPs。然后用western blot分析制备的HDFGFR4/CMMNPs(图1-2的C所示)。将存在于分子量为95kDa和125kDa区域的蛋白转移到硝化纤维素上进行进一步的免疫印迹检测。
结果显示FGFR4的western blot分析和半定量分析,FGFR4蛋白在HDFGFR4/CMMNPs中的表达明显高于FGFR4/CMMNPs。研究表明,基于HaloTag底物修饰的磁性纳米颗粒与Halo标记的细胞膜之间的位点特异性亲和力,可以实现HDFGFR4/CMMNPs上的高密度FGFR4。
共聚焦显微镜观察进一步证实了HDFGFR4/CMMNPs的成功制备,利用SA-APC对HDFGFR4/CMMNPs上的高fe Hacat细胞膜进行染色。FITC(绿色荧光染料,激发/发射光谱:488nm/520nm)染色MNPs核心。为了更好地观察HDFGFR4/CMMNPs的核壳结构,我们制备了大尺寸的MNPs。图1-2的D显示,SA-APC的红色荧光信号和FITC染色的核心的绿色荧光信号非常清楚地显示HDFGFR4/CMMNPs。相反,FGFR4/CMMNPs表现出微弱的红色荧光信号。这表明基于共价位点特异性细胞膜固定化的FGFR4在HDFGFR4/CMMNPs上的高密度表达。
3.2HDFGFR4/CMMNPs的制备与表征
为了表征HDFGFR4/CMMNPs的物理化学特征,采用透射电子显微镜(TEM)、扫描电子显微镜(SEM)、微红外光谱(MIR)、动态光散射(DLS)、X射线衍射(XRD)和振动样品磁强计(VSM)研究。HaloTag被构建到FGFR4的N端胞外结构域,通过亲和共价反应实现了HDFGFR4/CMMNPs表面由内向外的细胞膜包覆取向。在这里,CD47被用作特异性生物标志物来验证HDFGFR4/CMMNPs的细胞膜涂层取向。在HDFGFR4/CMMNPs溶液中加入细胞外或细胞内抗CD47溶液,然后加入金共轭抗兔IgG二抗溶液。显然,图2的A(c)和图2的A(d)显示了多个电子致密金颗粒聚集,表明FGFR4/CMMNPs表面同时存在内向外和外向外的细胞膜涂层取向。与FGFR4/CMMNPs不同的是,在HDFGFR4/CMMNPs中仅在图2的A(a)中观察到金颗粒的聚集,显示出亲和共价反应中由内而外的细胞膜涂层取向。
3.3HDFGFR4/CMMNPs的表征
为了验证HDFGFR4/CMMNPs的细胞膜表面,我们进行了SEM表征。显然,HDFGFR4/CMMNPs显示出来自单层细胞膜涂层的一致的粗糙表面,而裸露的MNPs显示出光滑的表面(图2的B)。对磁性特征也进行了研究,结果如图3所示磁滞现象无矫顽力和剩余力。在细胞膜涂覆过程中,饱和磁化强度降低,HDFGFR4/CMMNPs具有令人满意的超顺磁性,饱和磁化强度为27emu g-1。对HDFGFR4/CMMNPs的XRD谱图进行了表征,以测试其晶体结构。
如图4所示,HDFGFR4/CMMNPs显示了全部六个特征峰((220)、(311)、(400)、(422)、(511)和(440)),与JCPDS-international Center for Diffraction Data(JCPDS Card:19-629)文件中获得的Fe3O4的标准模式一致。结果表明,在整个制备过程中保留了Fe3O4的结构。
此外,通过共聚焦显微镜表征HDFGFR4/CMMNPs的细胞膜涂层。首先,在裸MNPs制备过程中,利用FITC制备HDFGFR4/CMMNPs。另一方面,Halo标记的FGFR4高表达细胞膜用细胞膜脂双分子层红色荧光染料DiI标记(激发/发射光谱:549nm/565nm)。结果如图2的C所示。很明显,绿色荧光和红色荧光信号重叠。结果表明,MNPs核心和halo标记的FGFR4高表达细胞膜具有很大的共定位。
综上所述,Halo标记的FGFR4高表达细胞膜被成功地覆盖在MNPs表面。DLS结果如图2的D所示。由于细胞膜包覆,HDFGFR4/CMMNPs的尺寸略大于裸MNPs。此外,在细胞膜涂覆过程中,HDFGFR4/CMMNPs的zeta电位从-10.54mV转移到-32.10mV,它也表现出与HFE Hacat细胞膜囊泡大致相当的表面电荷。
3.4吸附容量
HDFGFR4/CMMNPs的结合特性在实际应用中至关重要。因此,以FGFR4/CMMNPs为对照组,研究了HDFGFR4/CMMNPs在上述方法中的吸附能力。在整个实验过程中均使用BLU9931(一种FGFR4抑制剂)作为阳性药物。如图5的A所示,可以明显看出HDFGFR4/CMMNPs的吸附容量大于FGFR4/CMMNPs。HDFGFR4/CMMNPs的BLU9931吸附能力增强是高密度FGFR4受体的细胞膜包覆方法的好处,这导致HDFGFR4/CMMNPs的有效结合位点数量增加。结果表明,HDFGFR4/CMMNPs对BLU9931的吸附在BLU9931浓度为30~3000mg mL-1范围内迅速增加,随后逐渐达到352mg g-1的饱和平台。
吸附等温线是研究药物如何与制备的材料相互作用的有用工具。因此,得到的结合数据分别用Freundlich、Scatchard、Langmuir和Dubinin-Radushkevich等温线进行分析(表S1)。有趣的是,与以往的结果不同,HDFGFR4/CMMNPs的吸附平衡数据更符合Langmuir模型,相关系数最高(r2=0.9828)。而FGFR4/CMMNPs的吸附平衡数据更符合Freundlich模型,Freundlich模型是描述非均质表面吸附特性的常用模型。
HDFGFR4/CMMNPs与BLU9931的Langmuir等温线和Freundlich等温线如图5的B-C所示。Langmuir吸附等温线是一种流行的吸附模型,它假设表面位置均匀的单层吸附。Langmuir吸附模型的适配表明,HDFGFR4/CMMNPs表面存在均匀分布的FGFR4受体,进一步证明了本发明的优越性。
3.5吸附选择性
选择性是制备的生物材料的重要参数。为了评估HDFGFR4/CMMNPs的选择性,我们在HDFGFR4/CMMNPs筛选系统中纳入了五种药物。如图5的D所示,我们选择BLU55439和BLU9931作为细胞膜上FGFR4的阳性药物,随机选择洛伐他汀、阿司匹林和吉非替尼作为阴性对照药物,他们的结合位点均不是FGFR4。BLU9931对HDFGFR4/CMMNPs和FGFR4/CMMNPs的筛选回收率分别为92.56%和88.46%。另外,另一种阳性药物BLU554的筛选回收率分别为87.54%和85.49%,而两种吸附剂对其他三种阴性化合物的吸附量都很低。上述结果表明,HDFGFR4/CMMNPs对FGFR4阳性药物的选择性高于其他阴性药物。本实验证实了HDFGFR4/CMMNPs对FGFR4的生物活性化合物具有良好的选择性。
3.6吸附动力学
在实际应用中,HDFGFR4/CMMNPs的吸附动力学对筛选效率的贡献至关重要。HDFGFR4/CMMNPs的动态吸附性能如图5的E所示。可以明显看出,BLU9931在HDFGFR4/CMMNPs上的吸附在前6分钟迅速增加,然后趋于平稳,达到29.4mg g-1的吸附饱和,这与他们的静态吸附结果吻合得很好。FGFR4/CMMNPs表现出类似的趋势,但吸附量较低。高密度分布FGFR4受体的细胞膜包衣方法保证了HDFGFR4/CMMNPs的强结合能力。在实际应用中,HDFGFR4/CMMNPs的高传质能力有助于快速筛选生物活性化合物。
3.7筛选工艺参数优化
合适的洗脱过程对筛选结果至关重要。为了选择合适的洗脱液,我们考察了几种不同溶液的洗涤或洗脱能力。为了消除吸附剂的非特异性吸附分子,需要选择合适的洗涤溶液,在样品加载后洗脱非活性化合物。
结果如图5的F所示。用DMSO-50mmol L-1PBS(5:95,v/v)可以洗去BLU9931对MNPs的吸附,而在相同条件下,BLU9931对HDFGFR4/CMMNPs的亲和力不受影响。当洗涤液为DMSO-50mmol L-1PBS(15:85,v/v)时,两种材料均释放出BLU9931。因此,可以选择DMSO-50mmol L-1PBS(5:95,v/v)和DMSO-50mmol L-1PBS(15:85,v/v)分别作为合适的洗涤和洗脱液。
为了选择合适的吸附剂用量,我们将BLU9931溶液分别加载到不同量的HDFGFR4/CMMNPs和MNPs上。从图5的G可以看出,当吸附剂用量为10mg及以上时,HDFGFR4/CMMNPs对BLU9931的筛选效果较好。这表明当吸附剂剂量大于10mg时,可以提供足够的FGFR4结合位点。接下来,将BLU9931溶液加入不同洗涤或洗脱时间(1-15min)的HDFGFR4/CMMNPs和MNPs中,进一步研究洗脱时间。结果如图5的H所示。当洗涤时间大于5min时,BLU9931溶液达到最大回收率。为了使筛选过程不受天然产物中复杂基质的干扰,最终确定HDFGFR4/CMMNPs的投加量为10mg,洗脱时间为5min为最佳筛选条件。
3.8方法验证和应用
为了检验所建立方法的可靠性,对该方法的性能进行了评价。首先考察了BLU9931在0.01~200mg L-1的浓度范围内的线性关系,r值均大于0.9998,线性关系良好。方法的检出限为0.3×10-3μmL-1,灵敏度较好。此外,制备了6批用于筛选BLU9931的HDFGFR4/CMMNPs,其回收率的相对标准偏差(RSD)均小于10.2%,表明HDFGFR4/CMMNPs用于筛选天然产物中的生物活性化合物具有良好的重现性。上述结果表明,所建立的方法是可靠的。
磁性纳米材料的引入为筛选平台提供了一种快速有效的分离方法,可用于从复杂的天然产物中筛选生物活性化合物。因此,将制备的HDFGFR4/CMMNPs用于虎杖中潜在生物活性化合物的筛选。
图6的A为各步骤得到的总萃取液、上样液、洗涤液和洗脱液的色谱图。结果显示,加入总萃取液后,HDFGFR4/CMMNPs上保留了部分成分(A-b),洗涤步骤后大部分化合物被洗脱(A-c)。最终,在虎杖淋洗液中检测到三种主要化合物,推测其为潜在的生物活性化合物。对筛选的三种化合物进行TOFMS分析,分别为虎杖苷、大黄素和赤松素(图6的B所示)。因此,HDFGFR4/CMMNP可用于从复杂的天然产物中筛选生物活性化合物。
3.9药理作用
基于前期的实验结果,我们发现虎杖苷、大黄素和赤松素是FGFR4潜在的酪氨酸激酶抑制剂,可以很好地结合到酪氨酸激酶结构域。因此,我们进行了生物学实验来研究它们的潜在生物活性。
1.CCK8
采用CCK-8法验证虎杖苷、大黄素和赤松素对HepG2细胞的抑制作用。用BLU9931和培养基处理的细胞分别作为阳性对照组和阴性对照组。结果如图6的C和图7所示。这三种化合物对HepG2细胞的抑制作用随着药物浓度的增加而逐渐增强。此外,三种化合物对HepG2细胞的生长抑制活性呈剂量依赖性。对虎杖苷、赤松素和大黄素的半最大抑制浓度(IC50)分别为299.9、116.5和41.9μmol L-1。阳性药物BLU9931的IC50值为1.9μmol L-1,因此筛选到的潜在活性化合物需要进一步优化结构以获得更高的生物活性。因此,细胞活力测试表明,筛选的三种化合物对HepG2细胞具有显著的抑制活性。
2划痕试验
采用二维划痕实验研究了虎杖苷、赤松素和大黄素对HepG2细胞集体迁移的影响。不同化合物(包括BLU9931)处理HepG2细胞在不同时间间隔(0、24和48h)下的创面愈合情况见图6的D和图8、图9。显然,对照组的HepG2细胞在孵育48h后“伤口”基本愈合。相比之下,BLU9931处理24h和48h后,细胞迁移率分别为11.6%和12.6%。经三种化合物处理后,细胞迁移明显减少。虎杖苷、赤松素和大黄素处理48h后,细胞迁移率分别为19.0%、18.8%和22.3%,均显著低于对照组。结果表明,三种化合物均能阻止HepG2细胞的迁移,且与阳性药物的侵袭率基本相同。
3细胞凋亡测定
对HepG2细胞进行凋亡实验,进一步评价其诱导HepG2细胞凋亡的能力。图6的A和图7显示,这三种化合物均以剂量依赖的方式诱导细胞凋亡(图10、图11所示)。在200、400和800μmol L-1浓度下,虎杖苷诱导HepG2细胞的凋亡率分别为4.04%、6.3%和18.95%。在5、10和15μmol L-1浓度下,大黄素诱导HepG2细胞的凋亡率分别为16.4%、50.5%和78.86%。在30、120和240μmol L-1浓度下,赤松素诱导HepG2细胞的凋亡率分别为12.14%、9.71%和91.38%。此外,我们还研究了Blu9931处理的HepG2细胞。图6的A显示,在0.2、2和4μmol L-1浓度下,Blu9931诱导的HepG2细胞凋亡率分别为1.11%、2.05%和31.62%,这三种化合物均显示出良好的潜在抗肿瘤活性。
3.10分子对接研究
通过Sybyl-X 2.0对筛选的化合物进行分子对接研究,确定它们与FGFR4可能的结合模型。图6的B为虎杖苷、赤松素和大黄素和BLU9931的模拟结合模型。显然,虎杖苷、赤松素和大黄素与BLU9931的FGFR4酪氨酸激酶的ATP袋具有相似的结合构象。此外,虎杖苷和赤松素通过不同的氢键结合到FGFR4酪氨酸激酶结构域,包括Glu-622的侧链OH,其作用方式与阳性药物相似。因此,通过分子对接研究可以得出结论,筛选的三种化合物都是FGFR4的有效抑制剂。
以上给出的实施例是实现本发明较优的例子,本发明不限于上述实施例。本领域的技术人员根据本发明技术方案的技术特征所做出的任何非本质的添加、替换,均属于本发明的保护范围。
Claims (9)
1.一种基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,重组细胞膜上高表达目标膜受体,其N端融合表达Halo-tag蛋白标签;
重组细胞膜通过Halo-tag蛋白标签共价结合、反向包覆在氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒上。
2.如权利要求1所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述重组细胞膜上的目标膜受体为FGFR4受体;
重组细胞膜是反向包覆在氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒上,充分暴露受体的酪氨酸激酶区域。
3.如权利要求1所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述的Fe3O4@SiO2磁性纳米颗粒上还连接有链霉亲和素。
4.如权利要求1所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述的氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒通过以下途径制得:
1)通过法制备Fe3O4@SiO2磁性纳米粒子;
2)将Fe3O4@SiO2-NH2纳米颗粒羧基化得到Fe3O4@SiO2-COOH纳米颗粒;
3)将Fe3O4@SiO2-COOH纳米颗粒活化后与链霉亲和素共同孵育后,分离得到Fe3O4@SiO2-SA磁性纳米颗粒;
4)将Fe3O4@SiO2-SA磁性纳米颗粒与包含HaloTag配基的氯代烷烃修饰剂充分混合震荡,得到氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒。
5.如权利要求1所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述的重组细胞膜来自于慢病毒介导的含Halo-tag标签的FGFR4重组质粒转染HaCat细胞;
所述的含Halo-tag标签的FGFR4重组质粒是将含有Halo-tag标签的编码区插入FGFRR4的N端。
6.如权利要求5所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述的重组质粒为重组质粒LV-EFS>Halo tag/hFGFR4/3xEAAAK/EGFP-PGK>Puro。
7.如权利要求1所述的基于磁性纳米颗粒的受体高表达细胞膜药物筛选材料,其特征在于,所述重组细胞膜与氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒的共价结合为:
将5mg氯代烷烃修饰的Fe3O4@SiO2磁性纳米颗粒与1mL膜外区含Halo-tag标签的FGFR4重组细胞膜冰浴超声震室温下震荡混合2-4h,混合结束后使用甘氨酸溶液中和过量的生物素,再次用预冷的PBS溶液充分洗涤,得到FGFR4受体细胞膜包裹磁性纳米粒子。
8.权利要求1所述的高密度细胞膜受体包覆的细胞膜磁性纳米材料在筛选抗肿瘤药物中的应用。
9.如权利要求5所述的应用,其特征在于,是从包括虎杖在内的中药中筛选抗肿瘤的活性化合物。
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