CN116621901A - 一种基于糖代谢标记的小分子探针及其在提高铂类药物靶向性方面的应用 - Google Patents
一种基于糖代谢标记的小分子探针及其在提高铂类药物靶向性方面的应用 Download PDFInfo
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Abstract
本发明属于生物探针技术领域,尤其涉及一种基于糖代谢标记的小分子探针HBAPE‑Ac3ManNAz及其在提高铂类药物靶向性方面的应用。本发明提供的基于糖代谢标记的小分子探针HBAPE‑Ac3ManNAz,该探针作为代谢前体可以在体内外选择性标记肿瘤细胞或组织,并针对三阴性乳腺癌等缺乏特异性靶向受体等难治型肿瘤赋予新的靶标;合成与该探针正交反应的药效分子DBCO‑PEG8‑Oxp(IV)配合物,该配合物与标记在肿瘤细胞膜上的叠氮标签通过生物正交反应,提高铂类药物对肿瘤的靶向运输能力并减少毒副作用,为生物体内肿瘤的靶向治疗提供了新的理论依据。
Description
技术领域
本发明属于生物探针技术领域,尤其涉及一种基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz及其在提高铂类药物靶向性方面的应用。
背景技术
聚糖代谢标记(metabolic glycan labeling,MGL),已经成为一种简单但强大的工具,巧妙的借用了天然糖类的合成通路,将含有活性官能团(叠氮,炔基等)的非天然分子代谢标记到细胞表面,从而为肿瘤细胞人工引入化学受体。与常规肿瘤细胞表面自身含有的高表达受体相比,糖代谢标记因其可以活体标记并且由于高密度、低毒性、干扰小的特点使其为肿瘤细胞提供特异性治疗靶点成为可能。目前,已被开发出的四乙酰化N-叠氮乙酰甘露糖胺(tetra-acetylated N-azidoacetylmannosamine,Ac4ManNAz)尽管可以通过机体中唾液酸合成通路上的酶高效为肿瘤细胞表面糖链末端唾液酸引入叠氮基团,然后不可避免的是在正常的细胞中同样存在相同酶催化Ac4ManNAz的代谢标记,因此Ac4ManNAz在应用于肿瘤靶向治疗中难以实现对肿瘤组织的靶向性标记,体内肿瘤细胞选择性标记仍然是一个巨大的挑战。Bertozzi教授课题组于2000年首次报道了叠氮基团取代的N-acetylmannosamine(ManNAc)的类似物N-azidoacetylmannosamine(ManNAz),该探针分子可以经由唾液酸合成通路相应的酶转化成含叠氮唾液酸(SiaNAz)分子标记细胞膜表面的糖链结构上,从而在细胞膜表面人为地添加上了叠氮基团。与常规肿瘤细胞表面自身含有的高表达受体相比,糖代谢标记策略具有以下特点:(1)高密度:在细胞表面标记具有较高的覆盖率,约107个/细胞,比高表达受体多出10-1000倍;(2)低毒性:与抗体不同,引入的化学结构不易引发机体免疫应激等副反应;(3)干扰小:与抗体等大分子不同,非天然糖结构中引入的叠氮或者炔基等体积小不会干扰细胞间的识别与信息传递。基于以上优势,糖代谢标记结合生物正交反应策略可以靶向连接荧光团,对活细胞及活体水平对聚糖进行成像观测;也可以通过生物素与链霉亲和素的特异性结合实现对被标记的糖蛋白进行富集及组学分析;更为重要的是经典的反应基团为叠氮基和炔基不存在于哺乳动物体内,且需要一价铜离子催化,为了避免铜离子在活体内标记造成的毒性,Bertozzi课题组开发了叠氮基与二苯并环辛炔(dibenzocyclooctyne,DBCO)环张力促使的环加成(cyclooctyne-basedstrain-promoted alkyne-azide cycloaddition,SPAAC),也被称为无铜点击化学(copper-free click chemistry),可以在非铜离子催化条件下,高效共价连接如抗体、叶酸、多肽等形成糖基配体复合物,为糖链在体内代谢标记和肿瘤靶向治疗的应用研究提供了重要的保障。然而,近期研究发现大多数哺乳动物细胞同样存在着相同的酶催化传统的糖分子探针在高尔基体中通过翻译后修饰结合到细胞表面的聚糖上。因此,在不干扰其细胞代谢机制的情况下,使用ManNAz选择性地标记特定类型的细胞已被证明是困难的,特别在应用于肿瘤靶向治疗中难以实现对肿瘤组织的靶向性标记而导致药物脱靶分布对正常细胞及组织的损害。
活性氧(reactive oxygen species,ROS)是指氧衍生的活性小分子,包括过氧化氢,超氧负离子等,这些分子在细胞分化、细胞信号传递和适应性免疫等生物过程中发挥着重要作用。研究表明大多数肿瘤细胞中ROS含量偏高,可达10-100μM;而正常细胞内ROS含量通常为0.001-0.7μM。利用这一特点,已开发出ROS响应性载体,可以提高载体安全性的同时达到治疗疾病目的。已被开发出受ROS响应的4-(羟甲基)苯硼酸频哪醇酯(4-(Hydroxymethyl)benzeneboronic acid pinacol ester,HBAPE)不仅可以选择性靶向肿瘤细胞,还可以降低在正常细胞积累而导致的毒性,是药物化学研究中倍受欢迎的前药保护基团,常用于ROS调控的前药分子或者检测探针的保护基。
本发明针对正常体系和肿瘤***内ROS的含量差别,通过在Ac4ManNAz的1位引入4-羟基苯硼酸频哪醇酯结构(HBAPE),开发了一种依赖ROS调控以阻断在健康细胞中的代谢标记通路,进而可选择性化学标记和动态检测肿瘤细胞和肿瘤组织的新探针HBAPE-Ac3ManNAz,并合成与之正交反应的药效分子DBCO-PEG8-Oxp(IV),在实现铂类药物靶向性的同时增强抗肿瘤活性和减少毒副作用的产生。
发明内容
针对上述问题,本发明提供一种基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz,该探针作为代谢前体可以在体内外选择性标记肿瘤细胞或组织,并针对三阴性乳腺癌等缺乏特异性靶向受体等难治型肿瘤赋予新的靶标;合成与该探针正交反应的药效分子DBCO-PEG8-Oxp(IV)配合物,该配合物与标记在肿瘤细胞膜上的叠氮标签通过生物正交反应,提高铂类药物对肿瘤的靶向运输能力并减少毒副作用,为生物体内肿瘤的靶向治疗提供了新的理论依据。
为了实现上述目的,本发明所述的HBAPE-Ac3ManNAz结构式如下:
所述HBAPE-Ac3ManNAz的合成路线具体如下:
Reagents and conditions:(i)NaN3,NaOH,H2O,75℃,12h;(ii)N3CH2COOH,HOBT,EDC,MeOH,0℃,16h;(iii)Ac2O,Py,0℃,6h;(iv)4-Hydroxyphenylboronic acid pinacolester,Sc(OTf)3,1,2-C2H4Cl2,reflux,2h.
为了实现本发明另一个目的,本发明所述的与HBAPE-Ac3ManNAz探针正交反应的药效分子DBCO-PEG8-Oxp结构式如下:
所述DBCO-PEG8-Oxp(IV)的合成路线具体如下:
Reagents and conditions:(i)NH2OH.HCl,EtOH,Py,reflux,12h;(ii)PPA,125℃,1.5h;(iii)LiAlH4,Et2O,reflux,48h;(iv)methyl 4-chloro-4-oxobutyrate,Et3N,DCM,0℃,4h;(v)PyHBr3,DCM,Rt,24h;(vi)KOtBu,THF,-40℃,4h;(vii)LiOH,H2O,rt,12h;(viii)NH2-PEG8-NH2,HATU,DIPEA,DMF,rt,24h;(ix)H2O2,H2O,60℃,4h;(x)palmiticanhydride,DMSO,rt,7d;(xi)succinic anhydride,DMSO,rt,12h;(xii)HATU,DIPEA,DMF,rt,24h.
本发明所述的HBAPE-Ac3ManNAz作为代谢前体可以在体内外选择性标记肿瘤细胞或肿瘤组织。
优选的,本发明所述的HBAPE-Ac3ManNAz可以作为探针,在机体肿瘤内特异性标记,与化疗药物或者免疫治疗手段(包括基因治疗,抗原递送,siRNA等)协同作用,达到抗肿瘤的治疗目的。
优选的,所述的探针HBAPE-Ac3ManNAz可以与铂类药物协同抗肿瘤作用,其中HBAPE-Ac3ManNAz可以在肿瘤细胞膜特异性标记叠氮,提高铂类药物对肿瘤的靶向运输能力,并减少毒副作用。
所述的铂类药物可以是DBCO-PEG8-Oxp(IV)、顺铂等其他抗肿瘤作用的铂类药物。
优选的,所述的肿瘤包括但不限于缺乏特异性靶向受体等难治型肿瘤,例如三阴性乳腺癌。
本发明所述的DBCO-PEG8-Oxp(IV)配合物与探针HBAPE-Ac3ManNAz标记在肿瘤细胞膜上的叠氮标签通过生物正交反应,提高DBCO-PEG8-Oxp(IV)对肿瘤的靶向运输能力,并减少毒副作用,为生物体内肿瘤的靶向治疗提供了新的理论依据。
本发明的显著技术效果。
在本发明中,我们在已知探针Ac4ManNAz的1位引入4-羟基苯硼酸频哪醇酯结构,通过肿瘤细胞内高水平ROS可以提高探针对肿瘤细胞的选择性,抑制探针在低ROS含量的正常细胞中代谢活性,开发了一种依赖ROS调控的可选择性化学标记和动态检测肿瘤细胞和肿瘤组织的新探针HBAPE-Ac3ManNAz,尤其是对三阴性乳腺癌等缺乏有效靶标的肿瘤类型提供新的高密度肿瘤靶标分子,探针HBAPE-Ac3ManNAz具有安全高效且能在体内外特异性标记肿瘤细胞或组织的新型糖代谢标记分子探针。另外,我们结合生物正交反应策略,通过化学合成手段将铂类药物连接到DBCO上,利用生物正交反应与肿瘤细胞膜上叠氮基团进行结合,提高体内外肿瘤细胞及组织对铂类药物的摄入和铂类药物的靶向性,降低铂类药物的用量和对正常细胞的毒副作用,增强了药物抗肿瘤能力。糖代谢标记和生物正交反应的结合,为新型靶向抗乳腺癌铂类药物的开发提供理论依据,同时为为肿瘤的研究和靶向治疗方案的开发提供了新的参考。
附图说明
图1为HBAPE-Ac3ManNAz的细胞代谢标记表征。(a)HBAPE-Ac3ManNAz代谢标记的浓度梯度;(b)HBAPE-Ac3ManNAz代谢标记的时间梯度。
图2为HBAPE-Ac3ManNAz的细胞代谢标记效率受ROS调控。(a)HBAPE-Ac3ManNAz与不同浓度H2O2共培养的细胞标记水平变化;(b)HBAPE-Ac3ManNAz与不同浓度NAC共培养的细胞标记水平变化。
图3为HPLC检测HBAPE-Ac3ManNAz的水解效率受H2O2的调控。(a)HBAPE-Ac3ManNAz与不同浓度的H2O2共孵育3h;(b)HBAPE-Ac3ManNAz与相同浓度H2O2共孵育不同时间。HPLC进样条件:0-5min,5%甲醇;5-10min,15%甲醇;10-15min,20%甲醇。
图4为HBAPE-Ac3ManNAz在细胞膜上代谢标记的定位及效率分析。(a)HBAPE-Ac3ManNAz与Ac4ManNAz孵育24h的4T1细胞经DBCO-488、WGA、Hoechst 33342染色处理后的共聚焦成像(标尺:20μm);(b)4T1细胞与不同探针(200μM)一起孵育24小时,所收集的细胞并与DBCO-488偶联进行流式细胞术分析细胞膜表面荧光强弱。
图5为HBAPE-Ac3ManNAz小鼠体内肿瘤选择性标记。(a)Ac4ManNAz、HBAPE-Ac3ManNAz及HBAPE-Ac3ManNAz与NAC连用分别连续注射七天的小鼠经尾静脉注射DBCO-Cy524h后的全身荧光成像;(b)标记不同探针的小鼠尾静脉注射DBCO-Cy5 24小时后主要器官和肿瘤的荧光图像;(c)使用IVIS光谱成像***对标记不同探针的小鼠肿瘤组织的平均辐射效率进行量化(n=2)。n.s.表示无统计学差异,*P<0.05,**P<0.01,***P<0.001。
图6为铂类药物体外抗肿瘤活性评价。(a)阳性对照药奥沙利铂在4T1细胞中IC50值;(b)DBCO-PEG8-Oxp(IV)在4T1细胞中IC50值;(c)经HBAPE-Ac3ManNAz标记后的4T1细胞,DBCO-PEG8-Oxp的IC50值。
图7为ICP-MS检测4T1细胞对铂的摄入量。(a)经HBAPE-Ac3ManNAz标记24h前后的4T1细胞分别与1μM的奥沙利铂或DBCO-PEG8-Oxp(IV)孵育2h后细胞中铂的含量(n=3);(b)经HBAPE-Ac3ManNAz标记24h后的4T1细胞与不同浓度的DBCO-PEG8-Oxp(IV)孵育2h后细胞中铂的含量(n=3);(c)经HBAPE-Ac3ManNAz标记24h后的4T1细胞与1μMDBCO-PEG8-Oxp(IV)分别孵育不同时间后细胞中铂的含量(n=3)。n.s.表示无统计学差异,*P<0.05,**P<0.01,***P<0.001。
图8DBCO-PEG8-Oxp(IV)通过点击反应显著抑制4T1乳腺癌细胞的迁移能力。(a)伤口愈合实验探究HBAPE-Ac3ManNAz标记前后DBCO-PEG8-Oxp(IV)对4T1细胞迁移的抑制情况。左,图片,右,统计分析(n=3,标尺:50μm);(b)Transwell实验探究HBAPE-Ac3ManNAz标记前后DBCO-PEG8-Oxp(IV)对4T1细胞迁移的抑制情况。左,图片,右,统计分析(n=3,标尺:100μm);(c)标记前后DBCO-PEG8-Oxp(IV)对细胞生长迁移相关蛋白MMP-9和MMP-7表达影响。n.s表示无统计学差异,*P<0.05,**P<0.01,***P<0.001。
图9基于糖代谢标记,DBCO-PEG8-Oxp(IV)对4T1乳腺癌小鼠的抗肿瘤活性。(a)4T1乳腺癌小鼠荷瘤模型给药时间示意图;(b)治疗期间不同处理组小鼠体重变化曲线;(c)治疗期间不同处理组小鼠肿瘤生长体积变化曲线;(d)实验结束时的不同处理组小鼠肿瘤图片;(e)实验结束后不同处理组小鼠肿瘤的重量;(f)实验结束后不同处理组小鼠肿瘤的H&E染色(标尺:50μm);(g)实验结束后不同处理组小鼠各组织中铂含量分布(n=3)。n.s.表示无统计学差异,*P<0.05,**P<0.01,***P<0.001。
图10基于糖代谢标记,DBCO-PEG8-Oxp(IV)对4T1乳腺癌小鼠体内抗肿瘤转移活性。(a)4T1乳腺癌小鼠肺转移瘤模型给药时间示意图;(b)实验结束后不同处理组小鼠肺转移结节的代表性图像(n=5);(c)治疗期间不同处理组小鼠体重变化曲线(n=5);(d)实验结束后不同处理组小鼠的肺转移结节的统计数据(n=5);(e)实验结束后不同处理组小鼠肺组织的重量(n=5);(f)实验结束后不同处理组小鼠肺组织的H&E染色(标尺:50μm);(g)实验结束后不同处理组小鼠各组织中铂含量分布(n=3)。n.s.表示无统计学差异,*P<0.05,**P<0.01,***P<0.001。
具体实施方法
下面结合附图及具体实施方式对本发明技术方案做进一步详细描述。
实施例1。
本课题在Ac4ManNAz(结构式a)的基础上,将其1位乙酰基替换为受ROS调控的保护基4-(羟甲基)苯硼酸频哪醇酯(HBAPE),化学合成一种受ROS响应进而实现肿瘤选择性标记的新型糖代谢分子探针HBAPE-Ac4ManNAz(结构式b)。同时,将四价铂前药通过聚乙二醇(PEG)连接在DBCO上进一步合成水溶性DBCO-PEG8-Oxp(IV)(结构式c),所新合成的铂类药物在HBAPE-Ac4ManNAz靶向肿瘤标记基础上,通过高效的生物正交反应,将铂类药物靶向运输到肿瘤部位,从而增加其抗肿瘤活性和靶向性,减少其毒副作用。
1.HBAPE-Ac3ManNAz探针合成步骤如下:
(1)化合物1的合成
称取18g氯乙酸加入到250mL圆底烧瓶中,添加100mL蒸馏水中使其完全溶解,加入8g NaOH固体颗粒,待其搅拌均匀后逐渐加入15g NaN3,***N2球并设置反应温度为75℃,反应12小时后关闭加热装置至恢复室温,将圆底烧瓶中反应液倒入于含500mL冰水的烧杯中不断搅拌,配至20mL冰盐酸(15mL浓盐酸+5mL冰水)并逐渐加入烧杯中调节溶液pH至2-3,随后逐渐加入300mL乙酸乙酯进行萃取,重复三次,将收集到的乙酸乙酯用无水硫酸钠进行干燥,过滤并减压蒸除有机溶剂,得15.6g白色液体化合物1,产率为81%。1H NMR(300MHz,CDCl3)δ11.30(s,1H),4.02(s,2H).13C NMR(75MHz,CDCl3)δ174.74,50.06.
(2)化合物2的合成
称取5g盐酸-D-甘露糖胺加入到250mL圆底烧瓶中,添加100mL超干甲醇在室温下搅拌20min,随后依次加入3.6g的化合物1,9.6mL的Et3N,并将反应液移至冰水浴中搅拌30min,向反应液中加入2.2g HOBT和3.2g EDC,***氮气球并进行反应,16h后使用TCL监测反应情况,反应完全后减压蒸除有机溶剂,柱层析进行纯化得3.8g黄白色固体化合物2,产率为62%。
(3)化合物3的合成
称取2g的化合物2加入100mL的圆底烧瓶中,加入30mL的超干吡啶,移至冰水浴中不断搅拌,随后逐渐加入5mL的乙酸酐,***N2球并进行反应,6h后使用TCL监测反应情况,反应完全后减压蒸除有机溶剂,向250mL的分液漏斗中加入80mL 10%稀盐酸溶液(浓盐酸:蒸馏水=50mL:450mL)和120mL DCM进行萃取,收集下层有机溶剂并重复三次萃取操作,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得2.5g白色固体化合物3,产率为75%。1H NMR(300MHz,CDCl3)δ6.66(d,J=9.5Hz,1H),6.07(d,J=1.9Hz,1H),5.37(dd,J=10.2,4.2Hz,1H),5.31–5.16(m,1H),4.70–4.61(m,1H),4.26(dd,J=8.5,3.8Hz,1H),4.16(dd,J=12.1,2.3Hz,1H),4.11(s,2H),4.09(s,1H),2.21(s,3H),2.14(s,3H),2.09(s,3H),2.03(s,3H).13C NMR(75MHz,CDCl3)δ170.57,170.15,169.61,168.18,166.85,91.31,70.25,68.86,65.12,61.81,52.37,49.25,20.89,20.79,20.68,20.63.
(4)化合物4的合成
称取500mg的化合物3,1.37g的4-羟基苯硼酸频哪醇酯和345mg的三氟甲磺酸钪加入到100mL的圆底烧瓶中,并加入30mL的1,2-C2H4Cl2使其完全搅拌均匀,随后插上冷凝管,抽真空***N2球,升温到90℃反应两小时,TCL监测反应情况,反应完全后减压蒸除有机溶剂,向100mL的分液漏斗中加入30mL蒸馏水和50mL DCM进行萃取,收集下层有机溶剂并重复三次萃取操作,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得407mg白色固体化合物4,产率为58%。1H NMR(300MHz,CDCl3)δ7.85(d,J=7.9Hz,2H),7.39(d,J=7.8Hz,2H),6.56(d,J=9.4Hz,1H),5.41(dd,J=10.2,4.3Hz,1H),5.18(t,J=10.1Hz,1H),4.84(d,J=1.7Hz,1H),4.76(d,J=12.2Hz,1H),4.72–4.61(m,2H),4.26(dd,J=12.3,4.4Hz,1H),4.13(dd,J=6.4,1.9Hz,1H),4.06(d,J=10.4Hz,3H),2.17(s,3H),2.08(s,3H),2.01(s,3H),1.38(s,12H).13C NMR(75MHz,CDCl3)δ170.60,170.05,169.84,166.66,139.01,135.11,127.45,97.65,83.95,69.77,69.35,68.43,65.73,62.14,52.46,50.24,24.90,24.88,20.83,20.72,20.69。
2.正交反应的药效分子DBCO-PEG8-Oxp的合成步骤具体如下:
(1)化合物5的合成
分别称取20g的5-二苯并环庚烯酮和13.6g的盐酸羟胺加入到500mL圆底烧瓶中,依次加入60mL超干吡啶和240mL无水乙醇使其搅拌均匀,插上冷凝管,抽真空***N2球,升温到90℃回流反应12小时,TCL监测反应情况,反应完全后减压蒸除有机溶剂,加入350mL冰的5%稀盐酸在冰水浴中持续快速搅拌30min,搅拌过程中逐渐伴有白色固体析出,抽滤并用蒸馏水多次洗涤沉淀,晾干后用油泵彻底抽干,即得16g白色固体化合物5,产率为74%。1H NMR(300MHz,CDCl3)δ7.76–7.72(m,1H),7.68–7.64(m,1H),7.52–7.40(m,6H),6.96(d,J=2.1Hz,2H).13C NMR(75MHz,CDCl3)δ156.35,135.39,134.53,133.75,130.80,130.60,130.50,129.45,129.18,129.04,128.95,128.83,127.79,127.66.
(2)化合物6的合成
称取200mL聚磷酸加入到500mL圆底烧瓶中,将聚磷酸加热到125℃并在该温度下逐渐加入14g的化合物5并持续不断搅拌,反应过程中逐渐变为粘稠状黄色溶液,1.5h后关闭加热装置,待其恢复室温后将其倒入到含800mL冰水的烧杯并持续快速搅拌30min,搅拌过程中逐渐伴有灰褐色固体析出,抽滤并用蒸馏水多次洗涤沉淀,晾干后用油泵彻底抽干,即得11g灰褐色固体化合物6,产率为79%。1H NMR(300MHz,DMSO)δ9.89(s,1H),7.33–7.11(m,8H),7.01(d,J=11.7Hz,1H),6.90(d,J=11.4Hz,1H).13C NMR(75MHz,DMSO)δ172.20,136.76,136.59,134.92,133.91,133.11,130.66,129.44,129.39,128.54,128.26,128.23,127.87,126.85,126.68.
(3)化合物7的合成
分别称取6g的化合物6和2.6g的LiAlH4加入到500mL圆底烧瓶中,插上冷凝管,抽真空***N2球,随后向瓶中逐渐加入200mL超干***并不断搅拌,升温到45℃,期间不断补加***防止溶剂蒸干,48h后TCL监测反应情况,反应完全后将圆底烧瓶放入冰水浴中,逐渐向瓶中缓慢加入蒸馏水至无气体产生,随后加入200mL DCM进行萃取并重复三次,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得3g黄色固体化合物7,产率为53%。1H NMR(300MHz,CDCl3)δ7.37–7.22(m,4H),7.05(dd,J=7.8,1.6Hz,1H),6.96(ddd,J=8.4,7.1,1.6Hz,1H),6.69(td,J=7.6,1.2Hz,1H),6.62(d,J=13.2Hz,1H),6.54(dd,J=8.1,1.2Hz,1H),6.43(d,J=13.1Hz,1H),4.64(s,2H).13C NMR(75MHz,CDCl3)δ147.15,139.29,138.20,134.79,132.81,130.23,128.96,128.05,127.72,127.48,127.43,121.87,118.04,117.83,49.64.
(4)化合物8的合成
称取2.5g的化合物7加入到100mL圆底烧瓶中,依次加入40mL超干DCM和3.5mLEt3N,抽真空***N2球,随后将圆底烧瓶放入冰水浴中并不断搅拌,缓慢加入2.7mL丁二酸单甲酯酰氯,4h后TCL监测反应情况,反应完全后减压蒸除有机溶剂,并经柱层析进行纯化可得2.9g黄白色固体化合物8,产率为75%。1H NMR(300MHz,CDCl3)δ7.30(s,5H),7.21–7.13(m,3H),6.83(d,J=12.9Hz,1H),6.65(d,J=12.9Hz,1H),5.55(d,J=15.0Hz,1H),4.29(d,J=15.0Hz,1H),3.65(s,3H),2.68–2.58(m,1H),2.55–2.41(m,2H),2.11–1.99(m,1H).13CNMR(75MHz,CDCl3)δ173.48,170.93,140.58,136.53,135.89,134.65,132.71,131.91,130.95,130.24,128.63,128.33,128.10,127.36,127.03,54.56,51.70,29.63,29.10.
(5)化合物9的合成
称取2g的化合物8和2.5g PyHBr3加入到100mL圆底烧瓶中,添加40mL超干DCM,抽真空***N2球,室温下避光反应24h,TCL监测反应情况,反应完全后向瓶中加入30mL 10%稀盐酸溶液和20mL DCM并移至100mL的分液漏斗中进行萃取,收集下层有机溶剂并重复三次萃取操作,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得2.1g灰褐色固体化合物9,产率为70%。1H NMR(300MHz,CDCl3)δ7.76(d,J=9.0Hz,1H),7.26–7.02(m,6H),6.92(d,J=7.6Hz,1H),5.95(d,J=9.9Hz,1H),5.85(d,J=14.8Hz,1H),5.20(d,J=9.9Hz,1H),4.23(d,J=14.9Hz,1H),3.72(s,3H),2.96–2.83(m,1H),2.69–2.58(m,2H),2.58–2.46(m,1H).13C NMR(75MHz,CDCl3)δ173.60,172.05,138.35,137.10,136.98,132.82,130.85,130.76,130.71,129.70,129.54,129.02,128.95,128.64,60.15,55.63,52.61,51.81,30.72,29.29.
(6)化合物10的合成
称取2g的化合物9加入到100mL圆底烧瓶中,添加40mL超干THF,抽真空***N2球,将其放入-40℃下持续不断搅拌,10min后逐渐在该温度下缓慢加入8mL含1M叔丁醇钾的四氢呋喃溶液,2h后再补加3.2mL继续反应1h,TCL监测反应情况,反应完全后加入30mL蒸馏水和50mL DCM进行萃取,收集下层有机溶剂并重复三次萃取操作,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得800mg黄色油状化合物10,产率为60%。1H NMR(300MHz,CDCl3)δ7.72(d,J=8.8Hz,1H),7.60–7.49(m,1H),7.46–7.27(m,6H),5.20(d,J=13.8Hz,1H),3.67(d,J=9.6Hz,1H),3.59(s,3H),2.80–2.69(m,1H),2.66–2.58(m,1H),2.43–2.33(m,1H),1.99(dt,J=15.9,6.1Hz,1H).13C NMR(75MHz,CDCl3)δ173.31,171.70,151.48,148.04,132.31,129.32,128.55,128.30,128.16,127.76,127.13,125.49,123.16,122.70,114.98,107.75,55.50,51.65,29.56,29.11.
(7)化合物11的合成
称取500mg的化合物10加入到100mL圆底烧瓶中,添加20mL超干THF并持续搅拌,将75mg的LiOH溶于6mL的蒸馏水中随后加入于圆底烧瓶,室温下避光反应12h,TCL监测反应情况,反应完全后加入30mL蒸馏水和50mL DCM进行萃取,收集下层有机溶剂并重复三次萃取操作,将收集到的DCM用无水硫酸钠进行干燥,浓缩,柱层析进行纯化后可得300mg黄白色固体化合物11,产率为63%。1H NMR(300MHz,DMSO)δ12.05(s,1H),7.65(d,J=6.1Hz,1H),7.57–7.25(m,7H),5.04(d,J=14.1Hz,1H),3.63(d,J=14.0Hz,1H),2.68–2.52(m,1H),2.40–2.23(m,1H),2.27–2.11(m,1H),1.78(dt,J=16.3,6.4Hz,1H).13C NMR(75MHz,DMSO)δ174.03,171.22,151.93,148.89,132.85,130.08,129.39,128.66,128.48,128.14,127.29,125.63,123.02,122.02,114.76,108.51,55.41,29.74,29.45.
(8)化合物12的合成
称取150mg的化合物11加入到100mL圆底烧瓶中,添加15mL无水DMF,抽真空***N2球并持续搅拌使之完全溶解。随后称取280mg的HATU用6mL无水DMF溶解并加入瓶中,反应20min后,称取405mg的NH2-PEG8-NH2并用5mL无水DMF溶解,取0.41mL的DIPEA加入到溶解后的NH2-PEG8-NH2溶液中,10min后,向圆底烧瓶中加入NH2-PEG8-NH2与DIPEA的混合溶液,避光反应24小时,TLC监测反应,反应完全后减压蒸除有机溶剂,并经柱层析进行纯化可得214mg黄白色乳状化合物12,产率为65%。HRMS(ESI+)(m/z)calculated for C37H54N3O10[M+H]+700.3804,found 700.3790.
(9)化合物13的合成
称取1g的奥沙利铂于250mL的圆底烧瓶中,添加30mL蒸馏水并持续搅拌,随后逐滴滴加60mL的30%过氧化氢水溶液,60℃反应4小时待其完全溶解后,蒸除溶剂。在圆底烧瓶中加入100mL的蒸馏水继续搅拌,并将反应温度调至80℃,待瓶中反应液完全澄清后,停止加热,将烧瓶放于4℃冰箱中过夜,第二天会有大量白色针状结晶析出,将瓶中溶剂倒出,用油泵抽干结晶,即得900mg的白色固体化合物13,产率为83%。
(10)化合物14的合成
分别称取300mg的化合物13和310mg的棕榈酸酐于100mL的圆底烧瓶中,抽真空***N2球并向瓶中加入30mL无水DMSO溶剂后放于室温条件下搅拌7天,反应结束后将有机溶剂用油泵蒸发抽干,随后向瓶中加入50mL丙酮伴有大量白色固体析出,抽滤,并用丙酮反复清洗,晾干收集,即得350mg的白色固体化合物14,产率为75%。
(11)化合物15的合成
分别称取300mg的化合物14和100mg的丁二酸酐于100mL的圆底烧瓶中,加入加入30mL无水DMSO溶剂,抽真空***N2球反应12h,反应结束后减压蒸除有机溶剂,并经柱层析进行纯化可得210mg白色固体化合物15,产率为61%。
(12)化合物16的合成
称取100mg的化合物15加入到50mL圆底烧瓶中,添加10mL无水DMF,抽真空***N2球并持续搅拌使之完全溶解。随后称取75mg的HATU用3mL无水DMF溶解并加入瓶中,反应20min后,称取174mg的化合物12并用5mL无水DMF溶解,取0.19mL的DIPEA加入到溶解后的化合物12溶液中,10min后,向圆底烧瓶中加入化合物12与DIPEA的混合溶液,避光反应24小时,TLC监测反应情况,反应完全后减压蒸除有机溶剂,并经柱层析进行纯化可得89mg黄褐色乳状化合物16,产率为47%。1H NMR(300MHz,MeOD)δ7.45(d,J=2.5Hz,4H),7.39–7.20(m,4H),5.11(d,J=14.0Hz,1H),3.81–3.49(m,34H),3.50–3.29(m,6H),3.24(d,J=6.9Hz,4H),2.77–2.67(m,1H),2.67–2.45(m,2H),2.46–2.09(m,5H),1.97(dt,J=16.2,6.7Hz,2H),1.67–1.21(m,30H),0.91(t,J=5.8Hz,3H).13C NMR(75MHz,MeOD)δ173.40,173.17,172.96,172.52,151.31,148.11,132.14,129.26,128.68,128.29,127.84,127.55,126.77,125.11,122.99,122.24,114.23,107.53,69.95,69.80,69.13,55.31,54.44,38.97,31.74,30.52,30.02,29.96,29.44,29.16,22.41,17.36,15.92,13.19,11.87.
实施例2HBAPE-Ac3ManNAz的生物学评价
1.1活细胞的代谢标记
为了进一步探究HBAPE-Ac3ManNAz标记效率,我们将50、100、200、500μM不同浓度的HBAPE-Ac3ManNAz分别与4T1细胞孵育24小时来标记哺乳动物细胞中的蛋白,所裂解的蛋白与Biotin-PEG4-alkyne进行点击反应并通过Western Blot验证HBAPE-Ac3ManNAz对蛋白标记效率。结果如图1a所示,随着HBAPE-Ac3ManNAz浓度的增加,探针的代谢标记效果越来越强,当浓度达到200μM时,HBAPE-Ac3ManNAz可以对4T1细胞实现无细胞毒性的高效标记。
以此为参考,在该浓度下分别与4T1细胞孵育0、8、12、24、36、48h等不同时间长度,随后用同样方法进行蛋白标记效率验证,结果如图1b所示,随着HBAPE-Ac3ManNAz孵育时间的增加,其标记效果逐渐增强。在孵育时间达到24h时,HBAPE-Ac3ManNAz的细胞标记效果达到了一个理想的强度,随之延长其孵育时间时,36h标记效果更为强烈,随后再次延长但没有明显的差异。因此,从浓度和时间依赖性的实验结果综合来看,HBAPE-Ac3ManNAz在200μM浓度下对细胞标记24h时,可以实现对细胞的高效标记。
1.2HBAPE-Ac3ManNAz的细胞代谢标记受ROS调控
通过浓度、时间依赖性及细胞多样性实验对HBAPE-Ac3ManNAz的代谢标记进行了基本表征,可以证明新合成的HBAPE-Ac3ManNAz能够作为一个正常的糖代谢标记探针。基于此,通过Western Blot进一步探究HBAPE-Ac3ManNAz的细胞代谢标记是否受ROS调控。继续以4T1细胞为其代谢标记研究对象,在200μM HBAPE-Ac3ManNAz与4T1细胞共孵育24h的同时分别加入不同浓度的ROS激动剂H2O2溶液,后续经同样方法进行蛋白标记效率验证,结果如图2a所示,随着H2O2浓度不断增加,HBAPE-Ac3ManNAz的标记效果逐渐增强,表明HBAPE-Ac3ManNAz的细胞代谢标记对H2O2具有浓度依赖性。
同时,为了进一步说明HBAPE-Ac3ManNAz的细胞代谢标记受ROS调控,将不同浓度的ROS抑制剂N-乙酰-L-半胱氨酸(NAC)与200μMHBAPE-Ac3ManNAz共同孵育在4T1细胞中24h,其蛋白验证结果如图2b所示,当NAC浓度为0.5mM时,其标记效果显著降低,并随着NAC浓度不断增加而逐渐减弱,而浓度达到3mM时,其标记效果已基本完全抑制。以上结果可以得出糖代谢分子探针HBAPE-Ac3ManNAz在细胞的代谢标记过程中受ROS调控。
1.3体外HPLC检测ROS激动剂H2O2对HBAPE-Ac3ManNAz调控的变化
在HBAPE-Ac3ManNAz标记4T1细胞的时候通过添加ROS激动剂H2O2来改变探针的细胞代谢标记水平进而确定其标记过程受ROS调控。此外,为了进一步确定新合成的HBAPE-Ac3ManNAz中1位4-(羟甲基)苯硼酸频哪醇酯官能团的离去受ROS影响,我们采取了体外HPLC检测HBAPE-Ac3ManNAz在H2O2作用下1位4-(羟甲基)苯硼酸频哪醇酯官能团的水解效率。将HBAPE-Ac3ManNAz分别与不同浓度的H2O2在1.5mL EP管内共同孵育3h后进行HPLC检测,结果如图3a所示,随着H2O2浓度的增加,HBAPE-Ac3ManNAz的1位官能团的水解效率逐渐增加。
同时,将相同的浓度的HBAPE-Ac3ManNAz和H2O2共同孵育不同的时间进行HPLC检测,结果如图3b所示,随着孵育时间的增加,HBAPE-Ac3ManNAz的1位官能团的水解效率也逐渐增加。因此,以上两个HPLC检测结果更加佐证了新合成的HBAPE-Ac3ManNAz的水解效率受ROS激动剂H2O2的影响。
1.4共聚焦成像及流式细胞术对HBAPE-Ac3ManNAz在细胞标记中的定位和效率分析
在经典探针Ac4ManNAz的化学结构基础上进一步合成受ROS响应的HBAPE-Ac3ManNAz,因此在肿瘤细胞里高ROS含量作用下水解后其代谢标记机制与Ac4ManNAz理论上一致,均经唾液酸生物合成通路相应的酶转化成含叠氮唾液酸分子进而实现对细胞膜表面糖链末端唾液酸进行叠氮基团标记。为了验证此猜想,通过共聚焦成像及流式细胞术对Ac4ManNAz与HBAPE-Ac3ManNAz的细胞标记进行对比。将Ac4ManNAz与HBAPE-Ac3ManNAz分别以200μM的浓度与4T1细胞在共聚焦玻璃小皿中孵育24h,随后加入DBCO-488染料使叠氮与DBCO结合进而将探针标记位置处附带荧光,之后使用WGA染料对细胞膜表面糖蛋白染色和Hoechst 33342染料对细胞核染色作为对照,置于共聚焦显微镜下观察,结果如图4a所示,HBAPE-Ac3ManNAz与Ac4ManNAz的DBCO-488荧光区域一致,且与WGA染料染色区域重叠,说明新合成的HBAPE-Ac3ManNAz可以跟Ac4ManNAz代谢机制一样实现对细胞膜表面标记。此外,在荧光显微镜下观察可看出,HBAPE-Ac3ManNAz对细胞的标记水平略强于Ac4ManNAz,并且加入NAC共孵育后,HBAPE-Ac3ManNAz标记的荧光区域明显减弱,也进一步为HBAPE-Ac3ManNAz的代谢标记受ROS调控提供更充分的证据。
与此同时,为了验证HBAPE-Ac3ManNAz可以实现对细胞膜的高效标记,我们采用了流式细胞术对细胞膜表面荧光强度进行对比,将Ac36deoGlcNAz、Ac4GalNAz、Ac4ManNAz、HBAPE-Ac3ManNAz等多种不同类型探针分别与4T1细胞孵育24h,之后收集细胞用DBCO-488染料染色在流式机器上进行上机检测,结果如图4b所示,HBAPE-Ac3ManNAz在细胞膜上荧光标记效率达到了Ac4ManNAz的强度。两个实验的结果充分说明了新合成的HBAPE-Ac3ManNAz是一个高效标记细胞膜表面糖蛋白的代谢探针。
1.5HBAPE-Ac3ManNAz对小鼠体内肿瘤选择性标记
通过在细胞水平验证了HBAPE-Ac3ManNAz可以作为一个无毒、高效的糖代谢分子探针,因此想进一步验证HBAPE-Ac3ManNAz在小鼠体内荷瘤模型中对肿瘤细胞的标记效率。首先验证HBAPE-Ac3ManNAz的标记效率与给药次数的关系,将4T1肿瘤细胞通过皮下植入小鼠以构建荷瘤模型,待肿瘤成形后分别将不同组的小鼠分别腹腔连续注射七天Ac4ManNAz、HBAPE-Ac3ManNAz、HBAPE-Ac3ManNAz和NAC溶液(药物用量均为300mg/kg),随后经尾静脉注射100μg/μLDBCO-Cy5,24h后进行小动物活体成像并对肿瘤位置处荧光信号进行了活体成像比较,如图5a所示,HBAPE-Ac3ManNAz对肿瘤位置处的荧光信号强于Ac4ManNAz对肿瘤的标记效果,同时注射ROS抑制剂NAC后,HBAPE-Ac3ManNAz对肿瘤的标记效果明显减弱,说明新型探针HBAPE-Ac3ManNAz在体内肿瘤标记也受ROS调控。
同时,将各组小鼠解剖进行组织成像,如图5b-c所示,Ac4ManNAz在体内小鼠荷瘤模型中除肿瘤位置外,在肺及肾组织处均出现荧光信号,而新合成的HBAPE-Ac3ManNAz则极大地降低非肿瘤部位的代谢标记,增加了探针体内对肿瘤细胞的选择性标记,同时注射NAC的小鼠则抑制HBAPE-Ac3ManNAz的正常代谢标记过程,进而降低探针在肿瘤部位的表达,使其停留于肾脏处进行代谢而引起强的荧光信号。综合以上活体成像实验结果可以得出,与经典探针Ac4ManNAz相比,新合成的HBAPE-Ac3ManNAz体内代谢标记受ROS调控进而实现对肿瘤部位高选择性且高效标记。
实施例3。
一、基于HBAPE-Ac3ManNAz糖代谢标记,DBCO-PEG8-Oxp(IV)体内外抗肿瘤活性及靶向性研究
本发明以四价奥沙利铂为母核,在其两端的直立键中一端引入十六烷链,十六烷链修饰的铂类药物易于与人血清白蛋白(HAS)结合给药,药物在癌细胞中比在正常细胞中更优先积累;同时,直立键另一端引入DBCO,可以与提前经叠氮基团所标记的癌细胞发生点击反应来增强其肿瘤靶向运输能力。
1.体外抗肿瘤活性测试
通过化学合成得到目标化合物DBCO-PEG8-Oxp(IV),首先经MTT实验对其体外抗肿瘤活性进行评价,结果如图6所示,以阳性药二价奥沙利铂(图6a)作为对照,在4T1乳腺癌细胞中,化学合成的四价奥沙利铂配合物DBCO-PEG8-Oxp(IV)的IC50(图6b)低于二价铂,表明所合成的药物具有一定的抗肿瘤活性。与此同时,在经HBAPE-Ac3ManNAz标记24h后的4T1细胞中DBCO-PEG8-Oxp(IV)(图6c)的IC50值则达到了纳摩水平,肿瘤生长活性的抑制作用明显增强,说明点击反应介导的药物靶向运输可能在改善铂类药物靶向肿瘤能力和抗肿瘤活性上是一个很有前景的选择。
2.DBCO-PEG8-Oxp(IV)在乳腺癌细胞中铂的摄入量
药物在癌细胞的积累程度影响其抗肿瘤活性的强弱,铂类药物通过点击反应增强了药物的靶向能力并提高了细胞对药物的摄入量,因此进一步通过ICP-MS定量检测基于点击反应的DBCO-PEG8-Oxp(IV)在细胞中铂的含量。
我们将经HBAPE-Ac3ManNAz标记24h后和未经标记的4T1细胞分别加入终浓度1μM的DBCO-PEG8-Oxp(IV)孵育2h,同时以奥沙利铂为阳性药比较,孵育结束后所收集的细胞经硝酸酸化并采用ICP-MS检测,结果如图7a所示,未经标记的细胞分别与同浓度的奥沙利铂和DBCO-PEG8-Oxp(IV)孵育后,每2×105个细胞中铂含量并没有明显差异,而经HBAPE-Ac3ManNAz标记后的细胞中铂含量明显增高,几乎达到未标记的4到5倍,说明点击反应促进了细胞对铂类药物的摄入能力。此外,将0.5、1、2μM的DBCO-PEG8-Oxp(IV)分别孵育在经HBAPE-Ac3ManNAz标记24h后4T1细胞中2h,经ICP-MS检测,结果如图7b所示,每2×105个细胞中铂含量均明显高于对照组,但不同浓度之间细胞中铂含量并没有明显差异,我们猜测可能受制于孵育时间短的问题。另外,将1μM的DBCO-PEG8-Oxp(IV)分别孵育在HBAPE-Ac3ManNAz标记24h后4T1细胞中1h,12h,24h经ICP-MS检测,结果如图7c所示,每2×105个细胞中铂含量随着孵育时间的增加而增加,孵育24h后的细胞中铂含量则高达孵育1h时的4倍。以上实验结果表明基于糖代谢标记,DBCO-PEG8-Oxp(IV)通过点击反应促进了细胞对铂的摄入从而增加药物在肿瘤细胞中的积累。
3.DBCO-PEG8-Oxp(IV)对乳腺癌细胞迁移能力的影响
为了评价DBCO-PEG8-Oxp(IV)在点击反应介导下对乳腺癌细胞迁移的抑制作用。首先,通过伤口愈合实验检测DBCO-PEG8-Oxp(IV)对4T1乳腺癌细胞迁移能力的影响,将经HBAPE-Ac3ManNAz标记24h后和未经标记的4T1细胞分别加入终浓度1μM的DBCO-PEG8-Oxp(IV)进行孵育,同时以奥沙利铂为阳性药比较,于不同时间段在显微镜下进行观察拍照,结果如图8a所示,奥沙利铂和DBCO-PEG8-Oxp(IV)均能抑制4T1细胞的迁移,其中未经在HBAPE-Ac3ManNAz标记的细胞在12h时奥沙利铂和DBCO-PEG8-Oxp(IV)之间对细胞迁移的抑制作用有微弱的差异,但在24h后抑制作用基本无差异,然而经HBAPE-Ac3ManNAz标记的细胞,DBCO-PEG8-Oxp(IV)在12h及24h时其对细胞迁移的抑制作用均显著增强,实验结果说明DBCO-PEG8-Oxp(IV)通过点击反应促进了铂在肿瘤细胞上的积累进而增强对细胞迁移的抑制作用。
同时,为了进一步证明点击反应促进DBCO-PEG8-Oxp(IV)对细胞迁移的抑制作用,设计相同的实验组在Transwell小室中进行验证,结果如图8b所示,我们可以看出提前经过HBAPE-Ac3ManNAz标记24h的细胞,DBCO-PEG8-Oxp(IV)对细胞迁移能力的影响明显增强。
此外,我们还通过Western Blot探究在探针标记前后DBCO-PEG8-Oxp(IV)对细胞生长迁移过程中发挥重要作用的基质金属蛋白酶9(MMP-9)和基质金属蛋白酶7(MMP-7)的影响,结果如图8c所示,经HBAPE-Ac3ManNAz提前标记24h的细胞,DBCO-PEG8-Oxp(IV)均显著降低了MMP-9和MMP-7蛋白的表达,因此抑制了肿瘤细胞的生长迁移过程。以上实验结果充分说明DBCO-PEG8-Oxp(IV)通过点击反应显著增强了对乳腺癌细胞迁移能力的抑制作用。
二、基于HBAPE-Ac3ManNAz糖代谢标记,DBCO-PEG8-Oxp(IV)在小鼠体内抗肿瘤活性及抗转移效果评估
1.基于糖代谢标记,DBCO-PEG8-Oxp(IV)抑制乳腺癌细胞小鼠原位实体瘤生长实验
前期体外实验证明DBCO-PEG8-Oxp(IV)对经HBAPE-Ac3ManNAz所标记后的4T1乳腺癌细胞的杀伤作用显著提高,同时体内外也已经证明HBAPE-Ac3ManNAz对小鼠体内肿瘤组织具有强特异性且高效标记能力。因此我们将4T1乳腺癌细胞注射到Balb/C小鼠的腋下创建荷瘤模型,此实验分为PBS、Oxp.(2.46mg Pt/kg)、DBCO-PEG8-Oxp(IV)(2.46mg Pt/kg)、Ac4ManNAz+DBCO-PEG8-Oxp(IV)、HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)共五组,每组六只小鼠。给药周期如图9a所示,待小鼠平均瘤体积为50-100mm3,腹腔连续五天注射探针Ac4ManNAz或HBAPE-Ac3ManNAz,随后各组对应尾静脉注射相应药物Oxp.或DBCO-PEG8-Oxp(IV),铂类药物给药频率间隔两天注射一次,一共治疗三次,间隔期间经糖探针处理组别的小鼠每天对应补加探针。从接瘤开始每两天分别记录小鼠体重和肿瘤体积,给药结束后观察一天,取小鼠眼球血后并将其脱臼处死,随后取出肿瘤及各脏器组织进行拍照并记录肿瘤重量。给药周期中各组小鼠体重变化(图9b)趋于平稳,经Ac4ManNAz/HBAPE-Ac3ManNAz与DBCO-PEG8-Oxp(IV)联合的小鼠最后一天体重均不低于初始体重且最高不超过初始体重的20%,表明药物对小鼠无明显毒副作用。同时肿瘤体积变化曲线(图9c)显示,肿瘤生长的第11天,对照组小鼠肿瘤体积平均为920mm3,Oxp.组为711mm3、DBCO-PEG8-Oxp(IV)组为655mm3、Ac4ManNAz+DBCO-PEG8-Oxp(IV)组为290mm3,HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)组为185mm3,我们可以看出与对照组相比,奥沙利铂和DBCO-PEG8-Oxp(IV)均具有抑制肿瘤生长的作用,但抑制率分别为23%和29%,抑制效果并不明显且作用相差不大。当加入探针提前对肿瘤细胞进行标记时,DBCO-PEG8-Oxp(IV)对肿瘤的抑制作用显著增强,其中在Ac4ManNAz的标记下DBCO-PEG8-Oxp(IV)对肿瘤生长的抑制率达到了68%,而选用肿瘤选择性标记效果更好的HBAPE-Ac3ManNAz时DBCO-PEG8-Oxp(IV)对肿瘤生长的抑制率达到了80%。此外,对解剖的肿瘤组织进行拍照(图9d),随后并称重(图9e),使用探针标记时,DBCO-PEG8-Oxp(IV)对肿瘤的抑制作用更为明显,实体瘤重量更轻,与对照组相比,Ac4ManNAz+DBCO-PEG8-Oxp(IV)组小鼠实体瘤平均肿瘤约为对照组的21%,而HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)组小鼠实体瘤平均肿瘤约为对照组的14%。对各组肿瘤组织进行H&E染色,结果如图9f所示,对照组小鼠肿瘤组织的细胞数量显著增多,细胞核的体积增大,增殖能力强,而Ac4ManNAz/HBAPE-Ac3ManNAz与DBCO-PEG8-Oxp(IV)联合的小鼠肿瘤组织细胞核数量明显减少,并且也比单独使用奥沙利铂或DBCO-PEG8-Oxp(IV)组的小鼠的效果更为显著,表明在探针的作用下,DBCO-PEG8-Oxp(IV)对癌细胞增殖的抑制效果变强。
为了进一步验证DBCO-PEG8-Oxp(IV)通过点击反应增加了药物对肿瘤部位靶向运输能力,我们采用ICP-MS对各组小鼠组织的铂含量进行测定,结果如图9g所示,经Ac4ManNAz或HBAPE-Ac3ManNAz对肿瘤进行标记后,DBCO-PEG8-Oxp(IV)在肿瘤组织上铂积累量显著提高,Ac4ManNAz+DBCO-PEG8-Oxp(IV)组小鼠肿瘤上铂含量约为单独使用奥沙利铂或DBCO-PEG8-Oxp(IV)组小鼠的2倍,HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)组小鼠肿瘤上铂含量更高,可达3500ng Pt/g,同时从ICP-MS结果可以看出,与奥沙利铂相比,经过探针标记后,DBCO-PEG8-Oxp(IV)在体内肿瘤部位上积累更多,减少了肝肾等部位的积累,因此极大程度上减少对肝肾的损伤。并且使用HBAPE-Ac3ManNAz对肿瘤标记后DBCO-PEG8-Oxp(IV)的治疗效果要优于Ac4ManNAz对肿瘤的标记,说明HBAPE-Ac3ManNAz对肿瘤的标记具有更高的选择性从而增强了DBCO-PEG8-Oxp(IV)的靶向。以上实验结果表明在小鼠荷瘤模型中,基于糖代谢标记,DBCO-PEG8-Oxp(IV)通过点击反应提高了药物对肿瘤的靶向能力进而增强其抗肿瘤活性,同时减少铂在非肿瘤部位的堆积而引起的副作用。
2.基于糖代谢标记,DBCO-PEG8-Oxp(IV)抑制乳腺癌细胞小鼠肺转移实验
体外实验证明,基于HBAPE-Ac3ManNAz糖探针代谢标记,DBCO-PEG8-Oxp(IV)对乳腺癌细胞迁移能力具有显著抑制作用。为进一步验证,我们将鼠源4T1乳腺癌细胞采取尾静脉注射进Balb/C小鼠,创建小鼠肺转移模型。此实验分为PBS、Oxp.(2.46mg Pt/kg)、DBCO-PEG8-Oxp(IV)(2.46mg Pt/kg)、Ac4ManNAz+DBCO-PEG8-Oxp(IV)、HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)共五组,每组五只小鼠。给药周期如图10a所示,待接瘤后第五天时腹腔开始连续四天注射探针Ac4ManNAz或HBAPE-Ac3ManNAz,随后各组对应尾静脉注射相应药物Oxp.或DBCO-PEG8-Oxp(IV),铂类药物给药频率间隔两天注射一次,一共治疗三次,间隔期间经糖探针处理组别的小鼠每天对应补加探针。从接瘤开始每两天分别记录小鼠体重,给药结束后观察一天,取小鼠眼球血后并将其脱臼处死,随后取出各脏器组织,并将各处理组小鼠肺组织拍照(图10b)并记录肺重量及结节数。给药周期中各组小鼠体重变化(图10c)趋于平稳,各处理组小鼠体重变化不超过初始体重的20%。通过对肺组织观察发现(图10d),与对照组相比,DBCO-PEG8-Oxp(IV)和奥沙利铂处理的小鼠均具有微弱的乳腺癌转移能力的抑制作用,抑制率分别为27%和36%。当加入探针提前对肿瘤细胞进行标记时,DBCO-PEG8-Oxp(IV)对肿瘤转移的抑制作用显著增强,其中在Ac4ManNAz的标记下DBCO-PEG8-Oxp(IV)处理的小鼠肺结节降低了55%,而HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)处理的小鼠肺结节降低了84%。与此同时,我们对所剖出的肺组织进行称重,如图10e所示,充满结节的对照组的肺明显重于Ac4ManNAz+DBCO-PEG8-Oxp(IV)和HBAPE-Ac3ManNAz+DBCO-PEG8-Oxp(IV)所处理组的肺。随后对各处理组肺组织进行H&E染色,结果如图10f所示,对照组、奥沙利铂组及DBCO-PEG8-Oxp(IV)结节数过多的小鼠肺组织的细胞数量显著增多,细胞核的体积增大,增殖能力强,而Ac4ManNAz/HBAPE-Ac3ManNAz与DBCO-PEG8-Oxp(IV)联合的小鼠肺组织细胞核数量明显减少,说明在探针的作用下,DBCO-PEG8-Oxp(IV)对肿瘤转移的抑制效果显著变强。同时也可以发现使用HBAPE-Ac3ManNAz对肿瘤标记后DBCO-PEG8-Oxp(IV)的对肿瘤转移的抑制效果要优于Ac4ManNAz对肿瘤的标记,说明HBAPE-Ac3ManNAz对肿瘤的标记具有更高的选择性并增强了DBCO-PEG8-Oxp(IV)的靶向。
为了进一步验证DBCO-PEG8-Oxp(IV)通过点击反应增加了药物对肿瘤转移的抑制效果,我们采用ICP-MS对各组小鼠组织的铂含量进行测定,结果如图10g所示,经Ac4ManNAz或HBAPE-Ac3ManNAz对肿瘤进行标记后,DBCO-PEG8-Oxp(IV)在肺组织上铂积累量显著提高,而奥沙利铂处理的小鼠中铂主要分布于小鼠肝肾等部位。以上数据充分说明DBCO-PEG8-Oxp(IV)结合糖代谢标记可以靶向肿瘤细胞,进而抑制肿瘤的转移能力。
本发明设计的HBAPE-Ac3ManNAz是一种受ROS调控,具有安全高效且能在体内外特异性标记肿瘤细胞或组织的新型糖代谢标记分子探针;基于HBAPE-Ac3ManNAz糖代谢标记,DBCO-PEG8-Oxp(IV)提高了铂类药物对肿瘤细胞的靶向能力,增强了药物抗肿瘤活性和抗肿瘤转移能力,并且具有良好的生物安全性。
Claims (7)
1.一种基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz,其特征在于,所述的小分子探针HBAPE-Ac3ManNAz,其结构式如下:
2.如权利要求1所述的基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz在肿瘤内特异性标记并提高化疗药物的靶向性。
3.如权利要求2所述的基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz在肿瘤内特异性标记并提高化疗药物的靶向性,其特征在于,所述的所述的化疗药物为铂类药物。
4.如权利要求2所述的基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz在肿瘤内特异性标记并提高化疗药物的靶向性,其特征在于,所述的肿瘤包括但不限于缺乏特异性靶向受体。
5.如权利要求2所述的基于糖代谢标记的小分子探针HBAPE-Ac3ManNAz在肿瘤内特异性标记并提高化疗药物的靶向性,其特征在于,所述的化疗药物所述的探针HBAPE-Ac3ManNAz标记肿瘤细胞膜上的叠氮标签与化疗药物发生生物正交反应,提高化疗药物对肿瘤的靶向运输力。
6.一种与权利要求5所述的小分子探针HBAPE-Ac3ManNAz标记肿瘤细胞膜上的叠氮标签发生生物正交反应的抗肿瘤活性配合物DBCO-PEG8-Oxp,其特征在于,其结构式如下:
7.如权利要求6所述的抗肿瘤活性配合物DBCO-PEG8-Oxp,其特征在于,其特征在于,所述的DBCO-PEG8-Oxp作为药效分子与生物探针标记在肿瘤细胞膜上的叠氮标签发生生物正交反应,提高DBCO-PEG8-Oxp对肿瘤的靶向运输力。
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