CN112521388A - 一种苝酰亚胺衍生物及应用 - Google Patents
一种苝酰亚胺衍生物及应用 Download PDFInfo
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Abstract
本发明提供了一种苝酰亚胺衍生物及应用,属于生物医药技术领域,苝酰亚胺衍生物为结构式表示如下的化合物:
Description
技术领域
本发明属于生物医药领域,具体涉及一种苝酰亚胺衍生物及应用。
背景技术
肺癌病因和发病机制复杂,临床确诊者多为中晚期肺癌患者,主要采用以铂类药物化疗为主的治疗方案,但铂类化合物是肺癌的非优势分布药物,患者治疗过程易于产生耐药性和肝、肾毒性等副作用,对正常组织器官损伤较大。为改善肺癌化学治疗效果,可制备肺和细胞器优势分布药物。一方面增强药物在肺部富集,增加药物在肺的摄取量;另一方面通过药物损伤细胞器,高效杀伤肿瘤细胞,提高肺癌治疗效果。因此,设计和制备可同时实现肺和细胞器优势分布的药物是改善肺癌化学治疗的前提和基础。
苝酰亚胺衍生物是一类有机半导体分子,具有优异的化学和热学稳定性,广泛应用于光电和生物材料领域。此外,苝酰亚胺衍生物易于制备,可通过在苝四羧酸二酐苝湾位碳原子、酰亚胺位和羰基氧原子位引入多类取代基团,调控苝酰亚胺衍生物光电性质,实现其在生物医学领域的广泛应用。近些年,科研人员制备多种苝酰亚胺衍生物分子,实现光声成像引导下肿瘤光热或光动学治疗,但苝酰亚胺衍生物应用肿瘤化学治疗还有待研究。
肺表面活性物质是由II型肺泡上皮细胞合成和分泌的一种磷脂蛋白混合物,由70%~80%磷脂(主要成分为饱和二棕榈酰磷脂酰胆碱)、10%蛋白质和10%中性磷脂组成,分布于肺泡表面。基于肺表面活性物质主要为磷脂,可利用其磷酸根负离子和带正电荷药物分子间静电作用,驱动药物在肺部优势富集,实现药物肺优势分布。线粒体双层膜结构中含有丰富心磷脂,大量暴露的磷酸根负离子可通过正负电荷静电作用结合含正电荷药物,增加药物富集量,提高治疗效率。此外,线粒体跨膜电位内部带负电荷,可进一步增加含正电荷药物摄取,改善治疗效果。基于此,可在苝四羧酸二酐酰亚胺位引入季铵离子,增加其与II型肺泡上皮细胞和线粒体双层膜磷酸根负离子间静电作用,增加苝酰亚胺衍生物在肺和线粒体富集。然而,并不是所有带有苝酰亚胺衍生物都可实现肺优势分布。因此,需要引入取代基团实现苝酰亚胺衍生物在肺和线粒体优势分布。申请人在实践首次发现在苝湾位碳原子位引入卤素原子,减弱苝核电子云π-π共轭效应,通过调节苝酰亚胺衍生物分子内π-π共轭效应和季铵离子亲水效应实现其在肺和线粒体优势,通过调节线粒体功能实现优异抗肺癌性能,为制备高效、低毒、性能可调控的肺癌治疗药物提供理论依据和物质基础。
发明内容
一种苝酰亚胺衍生物,苝酰亚胺衍生物为结构通式表示如下的化合物:
上述苝酰亚胺衍生物在制备肺以及线粒体荧光探针中的应用。
上述苝酰亚胺衍生物在制备抗肺癌药物中的应用。
其中,所述抗肺癌药物为抑制肺癌细胞增殖的药物。
具体地,所述肺癌细胞为A549和/或H446细胞。
本发明针对肺和线粒体的结构特点,通过改变分子结构,提供一种可实现肺和线粒体优势分布,且具有优异抗肺癌性能苝酰亚胺衍生物诊疗剂。
附图说明
图 1 中:(a)PDIC-NC 和 PDI-NC结构式;(b)心、肝、脾、肺和肾组织荧光成像和(c)荧光强度量化结果;(d)心、肝、脾、肺和肾组织液荧光定量分析;(e)心、肝、脾、肺和肾组织冷冻切片荧光成像和(f)荧光强度量化结果;荧光图像标尺:50 µm;
图 2 中: A549细胞增殖率随(a)PDIC-NC和(b)PDI-NC浓度变化曲线;(c) H1299,B16, SW480, DLD-1, HeLa, 4T1 和 BEAS-2B细胞增殖率(细胞浓度每毫升5×104个,苝酰亚胺衍生物浓度为0.50 µg mL-1);(d)A549平板克隆细胞随PDIC-NC浓度变化图像和统计结果 和(e)H446平板克隆细胞随PDIC-NC浓度变化图像和统计结果。化合物与细胞孵育时间为24 h,实验重复三次,*p < 0.05; **p < 0.01; ***p < 0.001。
图3中:(a)A549和H446细胞线粒体共定位荧光图像和共定位系数,PDIC-NC化合物浓度:1 μg mL-1,线粒体探针Mitrotracker浓度:1 mM,孵育时间 3 h, 荧光图像标尺:20 µm; (b)A549和H446细胞透射电镜图像,PDIC-NC化合物浓度:1 μg mL-1,孵育时间 24 h,图像标尺:200 nm,黄色箭头指线粒体膜,蓝色箭头指细胞膜;(c)A549和H446粒体膜电位和(d)绿色荧光强度量化,PDIC-NC化合物浓度:1 μg mL-1,孵育时间 6 h,荧光图像标尺:20 µm;(e)A549和H446细胞三磷酸腺苷(ATP)量,PDIC-NC化合物浓度:2 μg mL-1,孵育时间 8 h;(f)A549和H446细胞活性氧荧光图像和(g)荧光强度量化,PDIC-NC化合物浓度:1 μg mL-1,孵育时间 6 h,荧光图像标尺:50 µm;(h)流式细胞术检测细胞凋亡图,PDIC-NC化合物浓度:0.5 μg mL-1,孵育时间 24 h。实验重复三次,*p < 0.05; **p < 0.01; ***p < 0.001。
图4 为 A549和H446细胞凋亡相关免疫印迹图和量化结果,检测蛋白为Cl.Caspase 9, Cl.Caspase 7, Cl.Caspase 3, Bax, Bcl2, PARP1, and Cl.PARP1.。
图5 为A549皮下瘤模型抗肿瘤效果:(a) A549皮下瘤模型治疗方案,尾静脉注射,注射剂量:2 mg Kg-1,7天/次,治疗周期28天;(b)A549皮下瘤图像;(c)A549皮下瘤肿瘤相对增长体积曲线;(d)肿瘤重量统计;(e)小鼠重量统计;(f)肺组织切片H&E、Ki67和TUNEL染色图像,图像标尺:50 μm;(g)Ki67和(h)TUNEL相对阳性率统计。数据统计为3或6小鼠标本,*p< 0.05; **p < 0.01; ***p < 0.001。
图6 为 肺癌原位诱发模型抗肿瘤效果:(a)模型设计实验方案,尾静脉注射,剂量:2 mg Kg-1,5天/次,治疗周期120天;(b)肺癌原位癌肺标本图像,白色圈为肺部肿瘤结节和(c)肿瘤结节数目统计;(d)肺组织切片H&E、PCNA和TUNEL染色图像,图像标尺:40 μm;(e)PCNA和(f)TUNEL相对阳性率统计。数据统计为3或6小鼠标本,*p < 0.05; **p < 0.01;***p < 0.001。
具体实施方式
以下结合附图和实施例对本发明的技术方案作进一步详细说明,但本发明的保护范围并不局限于此。
实施例1苝酰亚胺衍生物的制备和表征
PDIC-NC制备与表征。将N,N’-二甲基乙二胺(2.5 mmol)溶于N-甲基吡咯烷酮 (50mL),然后加入1,6,7,12-四氯-3,4,9,10-苝四羧酸二酐(1 mmol),氩气保护,于80 ℃反应24 h,冷却至室温,加入100 mL丙酮,室温搅拌2 h,抽滤,水洗至中性,加入 2 M HCl室温(20 mL)反应24 h,冷却静置,抽滤,干燥,溶于水透析24 h (透析袋规格: 500-1000Da),将透析液冷冻干燥 (-62 ℃, 24 h),得PDIC-NC。1H NMR (CF3COOD, 400 MHz) δ 3.29 (d, J = 6.2 Hz, 12 H), 3.87 (s, 4 H), 4.88 (t, J = 5.0 Hz, <1 H), 7.98 (s, 4 H),8.85 (s, 4 H). 13C NMR (100 MHz, CF3COOD): δ 167.32, 138.74, 136.30, 133.28,132.43, 125.30, 123.84, 123.83, 59.78, 46.10, 38.35; HRMS (MALDI-TOF): m/z:[M-H]+ calcd. for C32H25Cl4N4O4, 669.0607, found 669.0624.
PDI-NC根据文献报道制备。Xu, Z., Cheng, W., Guo, K., Yu, J., Shen, J.,Tang, J., … Yin, M. (2015). Molecular Size, Shape, and Electric Charges:Essential for Perylene Bisimide-Based DNA Intercalator to Localize in CellNuclei and Inhibit Cancer Cell Growth, ACS Applied Materials & Interfaces, 7 (18), 9784–9791.
下述各实验过程中苝酰亚胺衍生物PDI-NC和PDIC-NC溶液均为PDI-NC和PDIC-NC的水溶液。
实施例2苝酰亚胺衍生物器官分布
将正常老鼠分组(n = 3),通过尾静脉注射上述苝酰亚胺衍生物PDI-NC和PDIC-NC水溶液,取主要器官(心、肝、脾、肺、肾),通过:
(1)器官荧光成像检测。采用活体成像荧光成像系统,收集各主要器官荧光图像,量化荧光强度,分析化合物在主要器官分布情况(n = 3);
(2)器官荧光强度定量分析。取50-70 mg心、肝、脾、肺、肾,加入组织裂解液,提取组织液,开展荧光光谱检测,根据化合物标准曲线,定量分析化合物在心、肝、脾、肺、肾中分布(n = 3);
(3)器官组织冷冻切片检测。活体组织剥离后,制作器官冷冻切片,收集各器官组织切片荧光图像并量化分析,验证化合物在主要器官分布情况,结果如图1所示。
PDI-NC和PDIC-NC结构如图1a所示。评估了其在肺优势分布,PDIC-NC和PDI-NC尾静脉注射剂量2 mg Kg-1,注射频次,5天/次,注射时长120天,安乐死老鼠。如图1b,c所示,PDIC-NC经尾静脉注射至正常小鼠体内,通过器官成像检测,肺呈现较强荧光。荧光强度量化结果显示,肺组织中PDIC-NC含量分别是肝、心、肾、脾中含量的5、30、36、42倍。进一步称取等质量的肝、心、肾、脾和肺,粉碎并提取组织液进行荧光定量分析,肺组织中PDIC-NC含量是肝、心、肾、脾中含量的2.5‒6倍(图1d)。冷冻切片荧光图像也显示,肺组织中PDIC-NC含量是肝、心、肾、脾中含量的5倍(图1e, f),上述实验结果表明PDIC-NC可实现肺优势分布。
实施例3 苝酰亚胺衍生物抑制肺癌细胞增殖能力
采用非小细胞肺腺癌细胞A549和小细胞肺癌细胞H446作为肿瘤细胞模型,同时选择正常支气管上皮细胞BESA-2B和人正常肺成纤维细胞HFL-1作为细胞比较模型。所有细胞均采购于美国ATCC细胞库。待细胞达到80%左右的融合度后,用0.25%的胰酶消化传代培养,使用对数生长期细胞用于体外细胞活力实验。
(1)CCK-8测定测定苝酰亚胺衍生物抑制肿瘤细胞增殖能力
细胞消化后接种于96孔板中,每孔含100 μL细胞悬液(细胞浓度每毫升5×104个),置于细胞培养箱(5% CO2、37℃)培养12 h后设空白对照组、PBS对照组和化合物作用组研究细胞增殖。化合物作用组每孔(n = 3)加入20 μL(0-5 µg mL-1)苝酰亚胺衍生物,置于培养箱中继续孵育24 h,弃掉孔内培养基,采用PBS洗涤。洗涤后继续培养1 h,采用酶标仪测定450 nm处荧光强度值,根据下述公式计算细胞增殖率:
细胞增殖率(%) = OD(实验组-空白组)/ OD(对照组-空白组)× 100%
(2)平板克隆实验测定苝酰亚胺衍生物抑制肿瘤细胞增殖能力
取6孔板,每孔置入500个对数生长期细胞,置于细胞培养箱中(5% CO2、37℃)孵育,细胞贴壁后,加入20 μL(0-5 µg mL-1)苝酰亚胺衍生物,孵育4 h后弃去化合物,加入培养基培养14天,待细胞成单独细胞克隆时,采用甲醇固定,结晶紫染色30 min,弃去染液,干燥,显微镜观察并计数,评估苝酰亚胺衍生物抑制肿瘤细胞增殖能力。
以上结果如图2所示,采用CCK-8和细胞平板克隆实验研究了PDIC-NC和PDI-NC抑制肿瘤细胞生长能力。由图2所示,将不同浓度(0-5 µg mL-1)PDIC-NC与A549(8000个)和H446(5000个)细胞 (共同孵育24 h,CCK-8结果表明,当PDIC-NC浓度为0.62 µg mL-1时,约70%A549和50%H446细胞死亡,进一步增加PDIC-NC浓度至1.25 µg mL-1,A549和H446细胞抑制率分别为100%和78%(图2a,b)。根据CCK-8实验结果,PDIC-NC化合物A549半数抑制浓度(IC50)约为0.48 ± 0.02 µg mL-1,是临床一线抗肺药物顺铂的16-48% (表 1),表明PDIC-NC可高效抑制A549细胞生长。而PDI-NC的IC50约为2.43 ± 0.25 µg mL-1(A549),较PDIC-NC半抑制浓度提高5倍,表明苝湾氯原子可显著影响苝酰亚胺衍生物抑制肿瘤细胞增殖性能(图2a,b)。同时,申请人还检测PDIC-NC对其他肿瘤细胞和正常人支气管上皮细胞抑制性能。结果显示,当PDIC-NC浓度为0.50 µg mL-1,其他肿瘤细胞和正常人支气管上皮细胞的增殖率仍可保持到70%,表明PDIC-NC具有优异细胞选择性(图2c)。进一步通过细胞平板克隆实验验证PDIC-NC抑制A549和H446细胞增殖能力。如图2d, e所示,A549细胞抑制率约为99%(0.5 µg mL-1),H446细胞抑制率约为97%(1 µg mL-1),再次证明了PDIC-NC可高效抑制A549和H446肿瘤细胞增殖。
表 1 顺铂在A549细胞检测IC50
实施例4苝酰亚胺衍生物体抑制肿瘤细胞生长机制研究
(1)抑制细胞生长机制
(i)苝酰亚胺衍生物线粒体定位
根据前期预实验结果,取对数期生长期细胞 (5×105个),接种于35 mm2共聚焦培养皿,孵育24 h,移除培养液,加入苝酰亚胺衍生物溶液 (1 mL),继续孵育3 h。加入Mitotracker green荧光探针,继续孵育0.5 h,弃去染液,PBS洗涤3次。激光共聚焦显微镜观察,拍照,计算共定位系数。
(ii)细胞活性氧检测
将对数期细胞(5×105个)悬液置入12孔板,加入苝酰亚胺衍生物溶液(1 mL),孵育6 h,弃去培养基,PBS冲洗。每孔加入DCFH-DA (1 mL, 10 μM),继续孵育20 min,PBS缓冲液洗3次,激光共聚焦显微镜观察,拍照,量化荧光强度。
(iii)线粒体膜电位检测
将对数生长期细胞(5×105个)置于培养皿中,设空白对照组 (1 mL PBS)、CCCP阳性对照组 (1 mL, 1 mM)、化合物作用组 (1 mL),分别加入JC-1工作液(1 mL, 10 nM)后孵育20 min,JC-1缓冲液洗2次,激光共聚焦显微镜 (激发波长:488 nm) 观察,拍照,量化荧光强度。
(iv)ATP产生
将细胞置于六孔板(6000个/孔)中,加入40 μL 苝酰亚胺衍生物溶液,孵育4 h,除去培养液,每孔加入200 μL裂解液完全裂解细胞(于0 ℃条件下完成),离心裂解液(4 ℃,5min),置入黑壁透明底96孔板(20 μL/孔),加入100 μL ATP检测液,酶标仪检测,根据标准曲线计算ATP产生量。
(v)流式细胞仪检测肿瘤细胞凋亡
将细胞置于六孔板(10000个/孔),每孔加入苝酰亚胺衍生物20 μL,孵育4 h,收集细胞置入15 mL离心管离心(4 ℃,5min),冷PBS洗涤2次。每管加入500 μL buffer溶液,再依次加入10 μL V-APC和5 μL 7-AAD,于暗处混匀10 min,流式细胞仪检测。
(vi)透射电镜检测细胞亚结构形态
将细胞置于细胞培养瓶中,75%融合度时移除培养液,PBS洗涤2次,加5%戊二醛(pH 7.2)固定2 h。刮下单层细胞移入离心管,离心3 min(800 rpm),沉淀细胞用0.1 M磷酸漂洗液和PBS洗涤(3×15 min)后,1%锇酸固定1 h。然后用50%、70%、80%、95%、100%丙酮脱水,继而浸脂、包埋后聚合,切片(厚度70 nm),3%醋酸铀、枸橼酸铅染色,蒸馏水冲洗5次,透射电镜(120 kV)观察形态结构。
(2)蛋白信号通路检测
通过Western blot实验检测肿瘤细胞凋亡通路和内质网应激通路。
将化合物 (1 mL)与A549或H446细胞孵育后(两种细胞个数均为5×105个,化合物与A549孵育时,化合物浓度为0、0.1 µg mL-1、0.3 µg mL-1,化合物与H446孵育时,化合物浓度为0、0.2 µg mL-1、0.6 µg mL-1),除去培养液,冷 PBS 洗涤3次,加入100 μL蛋白裂解液和1% 蛋白酶抑制剂,裂解30 min(0 ℃),收集细胞置入离线管,超声细胞粉碎仪粉碎,4 ℃离心30 min (15000 rpm),取上清液(蛋白提取液)。随后,制胶并装配电泳装置,80 V恒压电泳,待mark基本分散开时,调整电压为100 V,当燃料到达胶底部时,停止电泳;剪裁PVDF膜并用甲醇活化,装配转膜装置,100 V恒压电转1.5 h。取膜TBST冲洗,牛奶封闭1 h,加入一抗,轻摇12 h,洗涤3次,加入相应二抗,45 min后TBST冲洗,显色拍照分析。
结果如图3和图4所示。如图3a所示,PDIC-NC在A549和H446线粒体中共定位系数分别为0.98和0.89,表明可实现线粒体优势分布。透射电镜图像显示,A549和H446细胞与PDIC-NC共同孵育24 h后,线粒体变短,发生肿胀,产生大量碎片(图3b),表明PDIC-NC可损伤线粒体结构。线粒体损伤将引发其膜电位发生变化,结果显示,A549和H446线粒体膜电位分别下降了5.2和4.6倍(图3c,d),表明线粒体氧化呼吸链受到破坏,其将导致三磷酸腺苷(ATP)量降低。进一步检测ATP量,结果表明A549和H446细胞ATP量分别减少了97%和78%(图3e),验证线粒体氧化呼吸链损伤。粒粒体氧化呼吸链损伤后将导致线粒体内膜中电子量增加,可以与氧气等发生电子交换,引发大量活性氧产生。通过荧光探针检测表明,A549和H446细胞活性氧产生量分别增加了3.5和6.5倍(图3f, g)。活性氧产生将诱发细胞凋亡,如流式细胞术检测所示,17%A549细胞和30%H446细胞发生凋亡(图3h)。随后申请人通过Western Blot检测与凋亡相关蛋白(图4),结果显示,Caspase 3剪切体在A549和H446细胞表达量分别增加了5倍和2.5倍。同时,促凋亡蛋白Bax表达量提高1.5倍,抗凋亡蛋白Bcl-2表达量下调了70%,表明细胞凋亡是导致细胞死亡重要原因。目前,以线粒体为研究对象,其他可能导致细胞死亡原因仍在研究中。
实施例5苝酰亚胺衍生物体内抗肿瘤性能
(1)动物模型构建
皮下瘤模型:根据前期预实验结果,选取16-18 g SPF级BALB/c裸鼠(适应于A549和H446细胞),在其右上肢注射100 μL细胞悬液(2.5×107个),跟踪肿瘤体积变化,肿瘤体积增加至60 mm3,尾静脉注射苝酰衍生物化合物。
小鼠肺原位癌模型:根据前期预实验结果,选BALB/c小鼠,每只小鼠腹腔注射体重乌拉坦(0.6 g/Kg/周),连续注射12周,小鼠肺癌形成。尾静脉注射苝酰亚胺衍生物,跟踪治疗效果。
(2)苝酰亚胺衍生物体内抗肿瘤性能评价
动物模型构建完成后,随机将模型老鼠设立对照组和实验组(8只/组),实验组裸鼠按预实验剂量尾静脉注射苝酰亚胺衍生物,对照组鼠尾静脉注射同量生理盐水。针对不同模型小鼠采取不同的处理措施:
皮下瘤模型:尾静脉给药7天/次,注射剂量:2 mg Kg-1,实验时间28天。老鼠体重和肿瘤体积记录3天/次,28天后安乐死小鼠,记录肿瘤重量,眼眶取血进行血液生化指标分析,取心、肝、脾、肺和肾,进行生物兼容性分析。
肺原位癌模型:尾静脉给药5天/次,注射剂量:2 mg Kg-1,实验时间120天,老鼠体重记录7天/次,120天后安乐死小鼠,记录肺重量和肺部结节数目,眼眶取血进行血液生化指标分析,取心、肝、脾、肺和肾,进行生物兼容性分析。
根据皮下瘤模型肿瘤质量,转移模型和原位模型收集肿瘤/体重比和肿瘤结节数目,评价苝酰亚胺衍生物体内抗肿瘤性能。
(3)生物安全性评估
收集小鼠眼眶动脉血,检测其生化指标,评价化合物生物安全性。收集小鼠肿瘤、心、肝、脾、肺、肾,做石蜡切片,苏木精和曙红 (H&E) 染色,分析评估化合物疗效和生物安全性;对小鼠肿瘤进行末端脱氧核苷酸转移酶介导的dUTP-生物素缺口末端标记 (TUNEL)染色,辅助分析化合物治疗效果;组织切片经梯度酒精脱水后采用3% H2O2 反应20 min,PBS洗涤3次,并置于抗原修复液中微波修复15 min,室温自然冷却后PBS洗涤3次,5%BSA封闭30min,滴加一抗(Ki67)置于4 ℃冰箱封闭过夜,PBS洗涤三次,滴加生物素化的二抗,室温孵育1 h,PBS洗涤后DAB溶液显色,荧光显微镜拍照评估Ki67染色。
以上结果如图5和图6所示,A549皮下瘤模型结果显示。 经28天治疗,同对照组相比(注射等量PBS),A549皮下瘤肿瘤体积明显缩小,肿瘤重量也显著较低,甚至有部分肿瘤完全消失(图5b-d)。PDIC-NC治疗组肿瘤体积较对照组肿瘤体积减少了75%,肿瘤重量减少了65%,可高效抑制肿瘤生长。苏木精和曙红 (H&E) 染色表明PDIC-NC可显著抑制肿瘤增殖,细胞核规则(图5f)。Ki67和TUNEL染色也表明,PDIC-NC可高效抑制A549肿瘤细胞的增殖,诱导其明显凋亡(图5f-g),证明PDIC-NC具有优异的抗肿瘤作用。肺癌原位模型结果表明(图6),PDIC-NC治疗组肺癌结节数量较对照组减少了55%(图6b,c)。苏木精和曙红 (H&E)染色表明PDIC-NC可显著抑制肿瘤增殖,细胞核尺寸规则(图6d)。PCNA和TUNEL染色也表明,PDIC-NC可高效抑制A549肿瘤细胞增殖,诱导其明显凋亡(图6d-f),证明PDIC-NC具有优异的抗肿瘤作用。此外,上述模型实验小鼠血液生化指标未见明显影响,具有较优异生物兼容性和较小副作用。上述A549皮下瘤模型和肺癌原位癌模型分别检测了PDIC-NC短期和长期治疗肺癌性能,为肺癌治疗化合物设计和制备提供实践基础。
上述研究结果表明,苝湾含氯原子的苝酰亚胺衍生物可实现肺优势分布,细胞实验表明PDIC-NC可高效抑制肿瘤细胞增殖;荷瘤动物实验结果表明,PDIC-NC具有优异抗肺癌性能。初步分子机制研究表明,PDIC-NC分子可定位于线粒体,通过产生活性氧杀死肿瘤细胞,为制备高效、低毒、性能可调控的肺癌治疗药物提供理论依据和物质基础。
以上所述是本发明的优选实施方案,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应该视为本发明的保护范围。
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