CN112521410B - 一种InCe6卟啉组装体、其制备方法及应用 - Google Patents
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Abstract
本发明公开一种InCe6卟啉组装体、其制备方法及应用,属于材料化学和生物学技术领域。本发明采用卟啉金属化结合微乳辅助的组装方法实现一种水溶性纳米光敏剂的制备。在本发明中,通过向卟啉中心引入金属,利用重原子较强的自旋轨道耦合作用,促进快速系间窜跃,进而提高光敏剂的光动力性能。同时,通过对组装过程的调控,获得了水分散性良好且尺寸均一的纳米颗粒。该纳米颗粒在低浓度(0.8μg/mL)、低光功率(655 nm,25 mW/cm2)下显示出良好的光动力性能。
Description
技术领域
本发明属于材料化学和生物学技术领域,具体涉及一种InCe6卟啉组装体、其制备方法及应用。
背景技术
恶性肿瘤(亦称癌症)是威胁人类健康的重大疾病之一。众所周知,传统的癌症治疗方法在抑制肿瘤生长的同时存在一些不足,如:治疗效果差、复发风险高、副作用较大。而光动力治疗作为一种新兴的、具有特定空间选择性且侵袭性较小的癌症治疗模式,已批准用于临床癌症治疗。光动力治疗(Photodynamic Therapy,PDT)是指在光激发下,滞留在病灶位置的光敏剂产生诱导细胞凋亡的活性氧物种,最终达到治疗效果。对于PDT而言,光敏剂、光源、组织氧作为三个必需因素,其中光敏剂的活性将决定活性氧产率,最终影响PDT效果。因此,光敏剂的设计与合成一直以来都是科研工作者的研究重点。
卟啉由于具有良好的生物源性和光化学反应活性,早在20世纪70年代就作为第一代光敏剂用于治疗皮肤表面的肿瘤,但由于卟啉单分子的大环共轭结构,卟啉光敏剂分子往往高度疏水,不能直接应用于体内抗肿瘤治疗。为了解决卟啉单分子的水溶性和体内滞留性,目前科研工作者主要通过包封/装载、化学键连接卟啉光敏剂的方式实现卟啉单分子的纳米化,但合成过程繁琐复杂、装载率低等问题也会对治疗效果产生一定的影响。
因此,如何通过简便高效的方法设计合成水分散性良好、光动力性能优异的卟啉光敏剂成为了目前亟待解决的问题。
发明内容
有鉴于此,本发明的目的在于提供一种InCe6卟啉组装体、其制备方法及应用,所述制备方法不仅简便高效,并且设计合成的卟啉纳米材料具有良好的水分散性,还表现出优异的光动力治疗效果。
为了实现上述发明目的,本发明提供以下技术方案:
一种InCe6卟啉组装体的制备方法,包括以下步骤:
(1)制备二氢卟吩e6的DMF/HCl溶液,备用;
(2)按照摩尔比nCe6:n乙酸铟=1:3~5的比例,制备乙酸铟的HCl溶液;
(3)将步骤(2)中乙酸铟的盐酸溶液加入步骤(1)的溶液中,80~100℃反应40 h ~60 h,反应结束后立即向反应溶液中加水,0~10℃静置10 h~15 h后,对溶液进行离心分离得到沉淀,将沉淀反复水洗至中性,冷冻干燥即可得到金属卟啉InCe6粉末;
(4)向金属卟啉InCe6粉末中先后加入相同体积的DMF、HCl溶液、氯仿,超声混合均匀后,移取下层金属卟啉InCe6/CHCl3溶液备用;
(5)将步骤(4)中分离出的金属卟啉InCe6/CHCl3溶液注入乳化剂溶液中,经超声造乳形成均匀的微乳液后,立即置于63℃~65℃水浴中搅拌去除溶液中的CHCl3,待完全挥发后将反应溶液移至冰水中迅速冷却至室温,将离心水洗后的沉淀分散在三次蒸馏水中,低温保存。
优选的,所述步骤(1)中,Ce6(二氢卟吩)的质量与溶液体积的比例为,mCe6:VDMF:VHCl=2.5 mg:1 mL:1 mL,盐酸为1N盐酸。
优选的,所述步骤(1)~(2)中,混合均匀的方式为搅拌:所述搅拌转速为500~800rpm,所述搅拌时间为30 min ~60 min。
优选的,所述步骤(2)中,乙酸铟的质量与HCl的体积比为,m乙酸铟:VHCl=7~12 mg:1mL,盐酸为1N盐酸,更优选为7.33 mg:1 mL,二氢卟吩e6和乙酸铟的摩尔比更优选为1:3。
优选的,所述步骤(3)中,离心和水洗金属卟啉沉淀的转速为13000 rpm/min ~20000 rpm/min,所述离心和水洗金属卟啉沉淀和的时间为30 min;反应温度优选90℃。
优选的,所述步骤(4)中,加入金属卟啉InCe6的质量与溶液体积的比例为,mInCe6:VDMF:VHCl:VCHCl3=5 mg:1 mL:1 mL:1 mL。
优选的,所述步骤(4)中超声功率为150 W;每加入一种溶剂,超声时间为20~30min;所述步骤(5)中,所述超声造乳的功率80 W;超声时间为1~2 min。
优选的,所述步骤(4)中,乳化剂溶液为十二烷基硫酸钠的水溶液;所述十二烷基硫酸钠水溶液的浓度为0.625~12.5 mM,金属卟啉InCe6/CHCl3溶液和十二烷基硫酸钠水溶液的体积比为1:10。
上述制备方法制得的InCe6卟啉组装体,为水分散性良好、尺寸均一的卟啉组装体纳米颗粒。
本发明还提供了所述的InCe6卟啉组装体在制备光动力治疗抗肿瘤药物中的应用。
进一步地,包括以下步骤:将InCe6卟啉组装体与NHS-PEG-NHS和cRGD混合,所述InCe6卟啉组装体、NHS-PEG-NHS与cRGD的质量比为1 mg:5 mg:5 mg,得到cRGD修饰的InCe6卟啉组装体。
一种cRGD修饰的InCe6纳米颗粒,由包括以下组分的原料制备得到:DSPE-PEG-NHS、InCe6卟啉组装体和cRGD,所述InCe6卟啉组装体、NHS-PEG-NHS与cRGD的质量比为1mg:5 mg:5 mg。
本发明得到金属卟啉InCe6粉末后,通过溶液转移的方法获取InCe6/CHCl3溶液,将InCe6的CHCl3溶液注入乳化剂的水溶液中超声造乳,形成“水包油”的微乳液,水浴挥发CHCl3后,立即转入冰水浴中冷却,即得到InCe6组装体溶液。
本发明提供了所述制备方法制备的金属卟啉组装体纳米颗粒,改善了卟啉单分子的水溶性及水分散性,且能通过EPR效应和靶向剂的连接实现主被动靶向,进而有效在肿瘤部位聚集。金属卟啉InCe6卟啉组装体纳米颗粒在655 nm激光照射下,具有良好的单线态氧产生效果。
在本发明中,所述cRGD修饰的InCe6纳米颗粒的制备方法包括以下步骤:
(1)将上述InCe6卟啉组装体溶液离心并水洗1次,收集沉淀,将沉淀分散于三次蒸馏水中,搅拌混匀后,用微量的三次蒸馏水溶解DSPE-PEG-NHS粉末并注入InCe6卟啉组装体的水溶液中,以650 rpm的转速,37℃恒温搅拌12 h后停止反应,再次离心并收集沉淀;
(2)将上述得到的沉淀分散于三次蒸馏水中超声分散,用微量的三次蒸馏水溶解cRGD粉末,在涡流振荡器上混匀15 s后快速注入上述沉淀的水溶液中,在37℃恒温摇床上,以500 rpm转速,反应12 h。反应结束后离心并水洗沉淀1次,即得到经cRGD修饰的卟啉组装体纳米颗粒。
在本发明中,所述离心和再次离心的转速独立为13000 rpm,所述离心和再次离心的时间为30 min。所述mInCe6 NPs:mDSPE-PEG-NHS:mcRGD=1 mg:5 mg:5 mg。
在本发明中所述卟啉组装体纳米颗粒应用于光动力治疗时,由于靶向剂的修饰,卟啉组装体纳米颗粒能主动靶向肿瘤细胞膜表面过表达的αvβ3整合素,实现在肿瘤部位的有效富集及细胞内吞,从而进一步提升光动力治疗的效果。
本发明提供了一种稳定、高效纳米光敏剂的制备方法,以二氢卟吩e6为组装基元,通过螯合中心金属赋予金属卟啉重原子效应,结合微乳辅助的组装方法,即在不同浓度和种类的乳化剂的辅助组装下,实现金属卟啉InCe6的纳米化。
本发明提供的InCe6纳米颗粒利用重原子较强的自旋轨道耦合作用,促进快速系间窜跃,进而增加激发三重态寿命,产生更多的活性氧,最终赋予卟啉纳米颗粒优异的光动力治疗效果。经实验证实:在低浓度(0.8 μg/mL)和低光功率密度(655 nm,25 mW/cm2)下,产生了近90%宫颈癌细胞的杀死率。
附图说明
图1为本发明所制备的金属卟啉单体InCe6和Ce6的紫外吸收谱图和红外光谱图;a为本发明所制备的金属卟啉单体InCe6和Ce6的紫外吸收谱图; b为本发明所制备的金属卟啉单体InCe6和Ce6的红外光谱图;
图2为本发明所制备的金属卟啉组装体InCe6 NPs的谱图; a为本发明所制备的金属卟啉组装体InCe6 NPs的SEM图; b为本发明所制备的金属卟啉组装体InCe6 NPs的粒径统计图; c为本发明所制备的金属卟啉组装体InCe6 NPs和金属卟啉单体InCe6的紫外吸收谱图; d为本发明所制备的金属卟啉组装体InCe6 NPs的元素mapping图;d图中第一张标尺是100 nm 后五张标尺是50 nm;
图3为实施例5水溶性单线态氧捕获剂ADPA检测InCe6 NPs的光动力性能结果图;其中, a为ADPA水溶液在655 nm激光器照射下的紫外吸收光谱图; b为本发明所制备的金属卟啉组装体InCe6 NPs与ADPA混合后在655 nm激光照射下的紫外吸收光谱图;以a及b中380 nm处ADPA吸收峰强度的变化为研究对象绘制的ADPA降解速率图如图c所示;
图4为InCe6 NPs连接靶向剂前后的红外光谱图;
图5为不同浓度的InCe6@cRGD与Hela细胞共同孵化后的激光共聚焦成像,各个图标尺均为50μm;
图6为不同实验条件下的细胞内活性氧检测;其中,0μg/mL 表示不加InCe6@cRGD且不进行光照,0μg/mL+L表示不加InCe6@cRGD,但是进行光照,其他依次类推;各个图标尺均为100μm;
图7为不同实验条件下的活死细胞双染检测,图中标尺:100μm;
图8为不同浓度的InCe6@cRGD孵化Hela细胞后的MTT实验结果。
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
将10 mg二氢卟吩e6(Ce6,C34H36N4O6)粉末溶解于5 mL DMF溶液,500 rpm搅拌30min后,再向溶液中加入5 mL 1 N HCl溶液,600 rpm搅拌1 h得到Ce6的DMF/HCl溶液。在电子天平上准确称取乙酸铟(C4H9InO6)粉末14.66 mg并溶解于2 mL 1N盐酸溶液中。
在搅拌条件下,将2 mL乙酸铟/HCl溶液注入Ce6的DMF/HCl溶液,700 rpm搅拌30min后,将混合溶液置于90℃烘箱中反应48 h。反应结束后立即将溶液倒入其3倍体积的蒸馏水中,放入4℃冰箱中静置12 h后,20000 rpm离心30 min并水洗至中性,-80℃冷冻干燥24 h即可得到金属卟啉InCe6粉末。
实施例2
将5 mg InCe6溶解于1mL DMF溶液中,150 W功率超声30 min后,加入1 mL 1 NHCl溶液,相同条件下超声混合均匀(150 W功率超声30 min),最后加入1 mL CHCl3溶液,超声混匀(150 W功率超声30 min后)后静置5 min,待溶液分层后移取下层1 mL InCe6/CHCl3溶液备用。
在1000 rpm搅拌下,将1 mL InCe6/CHCl3溶液快速注入10 mL 0.625 mM十二烷基硫酸钠的水溶液中,进行1 min、80 W的超声处理后,将混合溶液置于63℃的水浴锅,30 rpm搅拌30 min,使CHCl3完全挥发,而后将剩余溶液立即放入冰水浴中冷却至室温,离心水洗后将所得到的InCe6卟啉组装体分散于适量的三次蒸馏水并放置于- 4℃冰箱中低温保存备用。
实施例3
对实施例1中制备的金属卟啉InCe6分别进行紫外吸收(UV-vis,型号为AgilentTechnologies Cary 60)和红外光谱测试(IR,型号为布鲁克VERTEX 70)。如图1a所示,金属化后的卟啉分子在Q带的吸收峰数目减少且B带吸收峰发生红移至417 nm,Q带吸收峰蓝移至635 nm。图1b为Ce6金属化前后的红外光谱图,金属化后的卟啉中心吡咯环N-H特征振动峰(3295 cm-1、985 cm-1)消失,Ce6卟啉分子外的羧基特征振动峰(1703 cm-1、2967 cm-1)没有发生变化,证实金属成功进入卟啉中心吡咯环内,且外部羧基结构保持完好。
实施例4
对实施例2中离心分散好的InCe6卟啉组装体(InCe6 NPs)进行SEM、粒径统计、紫外吸收、TEM mapping测试.
图2a为InCe6 NPs的SEM图,如图所示的InCe6卟啉组装体的形貌为均一球形。
图2b为InCe6卟啉组装体的粒径统计图,InCe6 NPs的平均直径在80 nm左右。
图2c为InCe6自组装前后的紫外吸收谱图。InCe6经自组装后,组装体的吸收峰发生红移拓宽且在644 nm处有较强吸收峰。
图2d为InCe6 NPs的TEM mapping图,由图可知,C、N、O、In四种元素均匀分布在纳米颗粒表面。
实施例5
通过水溶性单线态氧捕获剂ADPA来检测InCe6 NPs的光动力性能。在单线态氧(1O2)的作用下,ADPA可被氧化为稳定的内过氧化物ADPAO2,而ADPAO2在330-400 nm处没有紫外吸收峰,所以通过紫外吸收谱图中ADPA在330-400 nm间特征吸收峰强度的变化来表征1O2的产生。
分别取1 mL 25 μg/mL InCe6 NPs溶液或三次蒸馏水于石英比色皿中,加入120 μL ADPA水溶液(单线态氧捕获剂),用光功率密度为0.12 W/cm2的655 nm激光器照射27min。每隔3 min测试ADPA在不同实验条件下的紫外吸收光谱,结果见图3a和图3b。
如图3a所示,330-400 nm间四个特征吸收峰没有变化,,说明ADPA具有良好的光稳定性,在0.12 W/cm2的655 nm激光器照射下,并不会引起捕获剂ADPA特征吸收峰的下降;结合图3b紫外吸收谱图的变化,证明了在相同激光照射下InCe6 NPs溶液产生了1O2最终引起了ADPA特征吸收峰的下降。为了更直观的证明InCe6 NPs材料的单线态氧产率,选取380 nm处ADPA的吸收峰强度变化为研究对象,绘制在0.12 W/cm2的655 nm激光器照射下ADPA的降解曲线,如图3c所示。ADPA水溶液在655 nm激光照射27 min后,ADPA几乎未变化,保持100%剩余量。而实验组InCe6 NPs在相同光照条件下,ADPA剩余量为13%,即ADPA降解量为87%,进一步证明了InCe6 NPs具有良好的单线态氧产率。
实施例6
在电子天平上准确称取1mg DSPE-PEG-NHS粉末溶解于微量三次蒸馏水中,超声混合均匀后,注入1 mL 200 μg/mL InCe6 NPs的水溶液中,以650 rpm的转速37℃恒温搅拌12h后停止反应,13000 rpm离心30 min,将得到的沉淀分散于1 mL三次蒸馏水中。随后将1mgcRGD粉末溶解于30 μL三次蒸馏水,在涡流振荡器上混匀15 s后快速加入上述沉淀的水溶液中,在37℃恒温摇床上,500 rpm反应12 h。反应结束后13000 rpm离心30 min,水洗沉淀1次,即得到经cRGD修饰的InCe6 NPs(InCe6@cRGD)。
图4为InCe6 NPs靶向修饰前后的红外光谱图。图4显示,1710 cm-1处羧基的振动吸收峰逐渐消失,同时1640 cm-1处出现明显的酰胺键特征吸收峰,证实靶向剂cRGD通过酰胺化反应成功修饰到纳米颗粒表面。
实施例7
采用标准的细胞培养方案,将500 μL含有约4×104个Hela细胞的完全培养基溶液加入玻底培养皿中,在5%CO2培养箱中贴壁培养24 h。使用完全培养基将离心水洗后的InCe6@cRGD沉淀稀释至不同的浓度(0 μg/mL、0.2 μg/mL、0.4 μg/mL、0.6 μg/mL)并与贴壁细胞共同孵化4 h后,使用PBS溶液洗去表面残留的的纳米材料后,观察并拍摄其激光共聚焦成像效果,结果见图5。
由图5可知,红色荧光位置基本与细胞内部重合,且随InCe6@cRGD浓度的增加,细胞内红色荧光强度增强,说明InCe6@cRGD能有效被Hela细胞摄取,且其摄取量与材料浓度呈正相关。
实施例8
采用标准的细胞培养方案,将500 μL含有约8×104个Hela细胞的完全培养基溶液加入玻底培养皿中,在5%CO2培养箱中贴壁培养24 h后,弃去上清,加入经完全培养基稀释为不同浓度的InCe6@cRGD(0 μg/mL、0.2 μg/mL、0.4 μg/mL、0.6 μg/mL),继续放置于5%CO2培养箱中培养4 h后,使用PBS缓冲溶液洗去多余的纳米材料。再加入500 μL DCFH-DA(活性氧检测试剂)孵化20 min后使用光功率25 mW/cm2的655 nm激光器光照4 min(对照组不进行光照处理)。光照处理完毕后,加入PBS缓冲液洗三次后用激光共聚焦成像进行活性氧的检测。
由图6可知,在光照条件下,InCe6@cRGD被细胞内吞后均能有效产生活性氧,且随InCe6@cRGD浓度的增加,绿色荧光增强,证实细胞内ROS含量增多。
实施例9
使用Calcein-AM/PI(活死细胞双染试剂)定性检测InCe6@cRGD NPs在655 nm激光照射下对Hela细胞的杀死效果。玻底培养皿培养Hela细胞,细胞数量大约8×104个,贴壁培养24 h后,与经完全培养基稀释的纳米材料(0 μg/mL、0.6 μg/mL InCe6@cRGD)共同孵化4h,将PBS+L组和InCe6@cRGD+L组进行4 min的光照处理(25 mW/cm2,655 nm),其余两组不加光。最后分别加入500 μL Calcein-AM/PI的Buffer溶液孵化25 min,PBS缓冲液洗三次后用激光共聚焦成像进行活死细胞的染色观察。
由图7可知,PBS组、PBS+L组(L表示光照)、InCe6@cRGD组均表现出绿色荧光,证实了在该光功率密度的激光照射下,并不会引起Hela细胞的凋亡并且InCe6@cRGD材料自身较低的暗毒性也不会引发细胞的凋亡,而实验组(InCe6@cRGD+L)却显示出大范围的红色荧光,证实InCe6@cRGD在655 nm激光照射下产生大量的活性氧,最终诱导Hela细胞凋亡。
实施例10
在96孔板中培养Hela细胞,大约3×104个/孔,贴壁培养24 h后,更换新鲜的培养基并加入不同浓度(0 μg/mL、0.2 μg/mL、0.4 μg/mL、0.6 μg/mL,0.8 μg/mL)的InCe6@cRGD溶液,继续在二氧化碳培养箱中与Hela细胞共同培养4 h,再使用PBS缓冲液洗去多余的纳米材料。将96孔板置于光功率密度为25 mW/cm2的655 nm激光下照射不同时间(0、2 min、4min、6min),弃去上清后更换新鲜的完全培养基继续培养12 h。第二天弃去上清溶液后,向每孔中加入100 μL经完全培养基稀释的CCK-8(Cell Counting Kit-8)溶液并孵化1 h后,使用酶标仪测试450 nm处的吸光度,计算Hela细胞的存活率。
由图8可知,InCe6@cRGD的暗毒性几乎可以忽略,随着光照时间和材料浓度的增加,其细胞存活率不断下降。综合分析发现,InCe6@cRGD在低浓度(0.8 μg/mL)、低功率(25mW/cm2)下,产生近90%的细胞杀死率,即在低浓度和低光功率下仍表现出优异的光动力治疗效果。
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。
Claims (10)
1.一种InCe6卟啉组装体的制备方法,其特征在于,包括以下步骤:
(1)制备二氢卟吩e6的DMF/HCl溶液,备用;
(2)按照摩尔比nCe6:n乙酸铟=1:3~5的比例,制备乙酸铟的HCl溶液;
(3)将步骤(2)中乙酸铟的盐酸溶液加入步骤(1)的溶液中,80~100℃反应40 h ~60 h,反应结束后立即向反应溶液中加水,0~10℃静置10 h~15 h后,对溶液进行离心分离得到沉淀,将沉淀反复水洗至中性,冷冻干燥即可得到金属卟啉InCe6粉末;
(4)向金属卟啉InCe6粉末中先后加入相同体积的DMF、HCl溶液、氯仿,超声混合均匀后,移取下层金属卟啉InCe6/CHCl3溶液备用;
(5)将步骤(4)中分离出的金属卟啉InCe6/CHCl3溶液注入乳化剂溶液中,经超声造乳形成均匀的微乳液后,立即置于63℃~65℃水浴中搅拌去除溶液中的CHCl3,待完全挥发后将反应溶液移至冰水中迅速冷却至室温,将离心水洗后的沉淀分散在水中,低温保存。
2.根据权利要求1所述InCe6卟啉组装体的制备方法,其特征在于,所述步骤(1)中,二氢卟吩e6的质量与溶液体积的比例为,mCe6:VDMF:V盐酸=2 mg:1 mL:1 mL,盐酸为1N盐酸。
3.根据权利要求1所述InCe6卟啉组装体的制备方法,其特征在于,所述步骤(2)中,乙酸铟的质量与盐酸的体积比为,m乙酸铟:VHCl=7~12 mg:1 mL,盐酸为1N盐酸。
4.根据权利要求1所述InCe6卟啉组装体的制备方法,其特征在于,所述步骤(4)中,加入金属卟啉InCe6的质量与溶剂体积的比例为,mInCe6:VDMF:VHCl:VCHCl3 =5 mg:1 mL:1 mL:1mL,HCl溶液浓度为1N。
5.根据权利要求1所述InCe6卟啉组装体的制备方法,其特征在于,所述步骤(4)中超声功率为150 W;每加入一种溶剂,超声时间为20~30 min;所述步骤(5)中,所述超声造乳的功率80 W;超声时间为1~2 min。
6.根据权利要求1所述InCe6卟啉组装体的制备方法,其特征在于,所述步骤(4)中,乳化剂溶液为十二烷基硫酸钠的水溶液;所述十二烷基硫酸钠水溶液的浓度为0.625~12.5mM,金属卟啉InCe6/CHCl3溶液和十二烷基硫酸钠水溶液的体积比为1:10。
7.权利要求1至6任一所述制备方法制得的InCe6卟啉组装体。
8.权利要求7所述的InCe6卟啉组装体在制备光动力治疗抗肿瘤药物中的应用。
9.根据权利要求8所述的应用,其特征在于,包括以下步骤:将InCe6卟啉组装体与NHS-PEG-NHS和cRGD混合,所述InCe6卟啉组装体、NHS-PEG-NHS与cRGD的质量比为1 mg:5 mg:5mg,得到cRGD修饰的InCe6卟啉组装体。
10.一种cRGD修饰的InCe6纳米颗粒,其特征在于,由包括以下组分的原料制备得到:DSPE-PEG-NHS、权利要求7所述的InCe6卟啉组装体和cRGD,所述InCe6卟啉组装体、NHS-PEG-NHS与cRGD的质量比为1 mg:5 mg:5 mg。
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