CN115054691A - 一种三元硫族金属纳米粒子及其制备方法和应用 - Google Patents
一种三元硫族金属纳米粒子及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及纳米药物技术领域,具体涉及一种三元硫族金属纳米粒子及其制备方法和应用。将铁源试剂、银源试剂和配体混合搅拌溶解;然后升温至80~95℃,维持10~30min;再升温至150~240℃,反应10~30min;加入有机溶剂沉淀产物,并以7500~9000rpm的转速离心5~15min即得。本发明方法简单,步骤少,可大量重复制备。该纳米药物可在近红外激光诱导下,快速高效将光能转换成热能,具有优异的光热转换性能。与此同时,在肿瘤微酸环境中释放铁离子,诱导肿瘤细胞发生铁死亡;近红外激光照射可加速铁离子的释放;最终实现肿瘤的光热/铁死亡协同高效治疗。
Description
技术领域
本发明涉及纳米药物技术领域,具体涉及一种三元硫族金属纳米粒子及其制备方法和应用。
技术背景
癌症是危害人类健康的重大疾病之一。手术治疗、放疗、化疗或这几种方法的联合使用是治疗癌症的主要手段。然后有些癌症如食管癌,鼻咽癌发病隐匿,恶性程度高,手术治疗后复发率高。而在癌症的放、化疗过程中,放射线或化疗药物杀死癌细胞的同时,可损伤骨髓的造血功能并降低患者的免疫力,存在较强的机体毒副作用,再加上肿瘤细胞的耐受作用,使得此类癌症经放、化疗后五年存活率也维持在较低水平。因此,开发一种安全性好、疗效显著的癌症治疗新方法尤为必要。
为了规避传统治疗方法带来的风险,基于纳米材料的光热疗法(Photothermaltherapy,PTT)在癌症的治疗中大显身手。PTT即利用光热材料在激光诱导下快速升温的特性,选择性的杀死肿瘤细胞,是一种局部疗法,具有选择性好、安全性高等优点。然而,在癌症的光热治疗过程中,往往会引起热休克蛋白(Heat shock protein,HSP)的高表达,HSP具有维持蛋白结构、抑制肿瘤凋亡的功能,HSP表达量的升高会削弱或完全抵消对癌症的光热治疗作用。
鉴于此,在对癌症进行单一光热治疗的同时,联合其他治疗手段,对癌症进行协同治疗,有望大幅提高癌症的治疗效果,具有重要的基础研究价值和指导意义。
铁死亡(Ferroptosis)是一种非凋亡形式的程序性细胞死亡,在此过程中,铁死亡引发剂直接或间接引起细胞内羟基自由基(·OH)蓄积而导致细胞死亡。由于·OH引起的脂质过氧化物(Lipid peroxidation,LPO)大量产生会严重地破坏细胞膜的结构和完整性,诱导细胞发生铁死亡可避免肿瘤细胞凋亡抑制或化疗药物耐受。因此,在光热作用导致细胞坏死或发生细胞凋亡的过程中,同时触发肿瘤细胞铁死亡,可以有更加高效地杀死肿瘤细胞,达到更好的抑瘤效果。
目前,基于光热/铁死亡的肿瘤治疗策略主要以光热材料(如金纳米棒)为基础,通过表面修饰装载铁离子或铁死亡诱导剂(如Erastin),构建纳米药物。并通过增加细胞内铁离子的浓度,提高·OH产生的速率和数量,促使细胞内脂质过氧化,进而引发铁死亡;或由铁死亡诱导剂直接引发铁死亡。但是,已报道的光热/铁死亡纳米药物大多存在结构复杂、构建步骤繁琐,铁离子或铁死亡诱导剂释放不可控等问题;因此,亟需设计构建一种结构简单构建简便的光热/铁死亡纳米药物,以达到高效触发光热作用/铁死亡杀伤肿瘤细胞的目的。
基于现有技术并结合上述分析,本申请的发明人拟提供一种具备优异的光热效应并可触发肿瘤细胞铁死亡的三元铁基硫族纳米药物及其构建方法,以用于肿瘤高效光热/铁死亡协同治疗。
发明内容
本发明的目的在于克服现有技术中的问题,提供一种三元硫族金属纳米粒子及其制备方法和应用。
本发明的目的通过以下技术方案予以实现:
一种三元硫族金属纳米粒子的制备方法,包括以下步骤:
S1.将铁源试剂、银源试剂和配体混合溶于有机溶剂1中,得到混合物;所述铁源试剂、银源试剂或配体中至少有一种含有硫元素;
S2.在惰性气体氛围中,25~35℃温度下,将步骤S1得到的混合物搅拌15~60min;然后升温至80~95℃,保温10~30min;再升温至150~240℃,反应10~30min;
S3.在步骤S2反应之后的体系中加入有机溶剂2,并以7500~9000rpm的转速离心5~15min,保留沉淀物,纯化,即得。
优选地,所述铁源试剂为二乙基二硫代氨基甲酸铁,配体为油酸和油胺。
优选地,所述铁源试剂为FeCl2,所述配体为油酸和十二硫醇。
所述银源试剂为为AgNO3,AgCl或二乙基二硫代氨基甲酸银(Ag(DDTC)),优选为Ag(DDTC)。
铁源试剂与银源试剂的摩尔比为1:1~2:1。配体与有机溶剂1的体积比为1:8。
优选地,有机溶剂1为油胺;有机溶剂2为乙醚和/或丙酮。
优选地,所述步骤S3中,纯化的步骤包括:将步骤S3得到的沉淀物重悬于甲苯或氯仿中;过滤之后洗涤,然后分散于正己烷中,然后加入浓度为0.001~0.01M的四氟硼酸亚硝的二氯甲烷溶液,搅拌6~12h,离心收集,即得。
洗涤过程所应用的溶剂为丙酮、乙醚或者乙醇中的一种。
所述方法制备得到的三元硫族金属纳米粒子。所述三元硫族金属纳米粒子的粒径为10~25nm。
所述三元硫族金属纳米粒子在制备抗癌药物中的应用。
所述应用,包括以下步骤:
S11.将所述三元硫族金属纳米粒子加入溶剂中,然后加入亲水化修饰的高分子,室温搅拌12~24h,离心收集产物;
S12.将步骤S11得到产物与肿瘤靶向分子反应,纯化后,即得抗药药物;
其中,步骤S11中溶剂为N,N-二甲基甲酰胺,二甲基亚砜或乙腈中的一种;所述亲水化修饰的高分子为DSPE-PEG2000或DSPE-PEG2000-R,R为氨基、羧基或巯基。
进一步,步骤S12中反应条件为3~5℃,反应10~14h。纯化方法为透析法,透析袋的截留分子量为5000~8000Da,透析时间为48~72h,透析液采用去离子水。
所述肿瘤靶向分子叶酸或cRGD。
与现有技术相比,本发明具有以下技术效果:
(1)本发明提供一种用于肿瘤光热/铁死亡协同治疗的纳米药物,以三元硫族金属纳米粒子为基础,进行亲水化修饰和生物功能化,丰富了光热/铁死亡协同治疗药物的种类。
(2)本发明通过化学合成一次性制备获得多功能的纳米药物,省去铁死亡诱导剂的装载等步骤,方法简便,操作简单,且涉及的设备及原料均廉价易得,可重复大量制备。
(3)本发明可通过控制近红外激光的强度及光照时间,精准调控纳米药物升温幅度,以及铁离子的释放,从而使肿瘤光热/铁死亡治疗协同增效,以达到最优的精准治疗效果。
(4)本发明的纳米药物,具有光声成像功能,在肿瘤诊断与精准治疗中具有较好的应用前景。
附图说明
图1为实施例1中,三元硫族金属纳米粒子的微观形貌。
图2为实施例2中,纳米药物在水溶液中的粒径分布。
图3为实施例3中,纳米药物在近红外激光诱导下的体外升温。
图4为实施例4中,纳米药物体外抗肿瘤性能检测结果。
图5为实施例5中,纳米药物体内肿瘤光热/铁死亡协同治疗结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例和对比例将对本发明的技术方案进行详细的描述。显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式,都属于本发明所保护的范围。
除特殊说明,本实施例中所用的设备均为常规实验设备,所用的材料、试剂若无特殊说明均为市售得到,无特殊说明的实验方法也为常规实验方法。
实施例1
三元硫族金属纳米粒子的化学合成,具体步骤如下:
(1)Fe(DDTC)3的合成:称量取六水氯化铁0.27g、三水合二乙基二硫代氨基甲酸钠0.68g分别溶解于100mL去离子水中。将两种溶液混合,室温搅拌1小时。收集白色沉淀,并用去离子水洗涤数次,置60℃真空干燥箱中干燥即制得Fe(DDTC)3。以同样的方法,制得Ag(DDTC)。
(2)称量取50.06mg的Fe(DDTC)3、25.61mg的Ag(DDTC),置50mL两口烧瓶中,加入油酸2mL、油胺16mL。将烧瓶置磁力搅拌电热套中,通氩气,排尽反应体系中的空气。
(3)将反应体系升温至95℃,维持10分钟。
(4)将反应体系快速升温至180℃,反应30分钟。冷却至45℃。
(5)往反应体系中加入30mL丙酮,混匀。8000rpm×10分钟离心,弃上清,收集产物,重新分散于甲苯中,即制得含铁的三元硫族金属纳米粒子。
该三元硫族金属纳米粒子在透射电镜下的微观形貌如图1所示。从图1中可以看出,该纳米粒子形貌比较规则,为球型。在有机溶剂中分散性好,为单分散纳米粒子。经测量后其粒径为18.0±3.4nm,粒径分布较集中。
实施例2
三元硫族金属纳米粒子的亲水化修饰和生物功能化,具体步骤如下:
(1)取25mg的实施例1中制得的三元硫族金属纳米粒子,加入10mL丙酮,洗涤2次,重新分散于5mL正己烷中(~5mg/mL)。
(2)称量取0.01mmol的NOBF4溶剂,溶解于5mL无水二氯甲烷中,浓度为2mM。
(3)将该三元硫族金属纳米粒子和NOBF4溶液混匀,室温搅拌12h。8000rpm×10分钟离心收集沉淀,加入0.5mL DMF分散。
(4)加入10mL甲苯/正己烷(体积比1:1),对分散于DMF中的纳米粒子进行纯化洗涤。8000rpm×10分钟离心收集沉淀,分散于5mL DMF中。
(5)称量取75mg DSPE-PEG2000-NH2,溶解于2.5mL DMF中(30mg/mL)。将分散于DMF中的纳米粒子和DSPE-PEG2000-NH2溶液混合,室温搅拌12h。
(6)加入过量丙酮沉淀产物,8000rpm×10分钟离心收集纳米粒子。加入2.5mL去离子水,超声即得表面修饰有-NH2水溶液中分散均匀的纳米粒子(~10mg/mL)。
(7)肿瘤靶向分子叶酸(Folic acid,FA)处理:取10mg的FA,超声溶解于1mL二甲基亚砜中,然后8000rpm×10分钟离心收集取上清。加入EDC(N-(3-dimethyl aminopropyl)-N′-ethylcarbodiimide hydrochloride)和NHS(N-hydroxy succinamide)活化叶酸的羧基,摩尔比为FA:EDC:NHS=10:1:1,室温反应2小时。
(8)将表面修饰有-NH2的纳米粒子和(7)中处理后的FA溶液混合,4℃搅拌12小时。然后8000rpm×10分钟离心收集取产物,即制得对肿瘤表面叶酸受体(FRα)具备靶向能力的纳米药物。
分散于水溶液中的纳米药物的水力直径如图2所示,纳米药物的粒径分布比较集中,说明在水溶液中的分散性良好,未发生团聚沉淀。经测量,其粒径为66.2±23.7nm。
实施例3
具有诱导肿瘤细胞光热治疗/铁死亡的纳米药物体外升温检测,具体步骤如下:
(1)稀释浓度为0.02、0.05、0.1、0.2、0.5mg/mL的纳米药物溶液。取100μL该纳米药物置微型玻璃管中,开口处用封口膜封口防止水分蒸发热量散失,中介仅留一个直径为1mm的热电偶测温仪插入的小孔。
(2)将起始温度调整为25℃,用功率为1W/cm2的808nm激光分别照射30秒、1、2、3、5、7、10分钟,照射完毕后立即测定样品温度。
(3)每个浓度的纳米药物溶液,每个激光照射事件段测定三次,求平均值。
如图3所示,为不同浓度的纳米药物在808纳米激光照射下的升温幅度,从图3中,随着纳米粒子浓度增加,其升温幅度逐渐增大,说明其升温具有浓度依耐性。同时,同一浓度的纳米粒子溶液,激光照射时间越长,其吸收的热量越多,升温幅度也越大。但高浓度的纳米粒子(0.5mg/mL),激光照射7min后,其温度会维持在平台期,不再随着照射时间的延长继续升高。浓度为0.2mg/mL的纳米粒子溶液,激光照射10min后,其升温幅度达到50.7摄氏度,同等条件下的水溶液,仅升温7.3摄氏度。该结果表明,我们构建的纳米药物具备优异的光热转换能力,可高效的将光能转换成热能。
实施例4
本发明述及的纳米药物用于肿瘤细胞体外光热/铁死亡治疗,具体步骤如下:
(1)该实施例以乳腺癌4T1细胞为例,但不限于此。该部分实验分别用Hoechst33342/PI荧光双染色法及CCK-8法对治疗后的肿瘤细胞活性进行定性、定量分析。
(2)将乳腺癌4T1细胞传代至96孔板中,每孔约5000个细胞,过夜培养。
(3)实验共分为4组,分别为PBS对照组(每孔加100μL PBS)、PBS+激光照射组(每孔加100μL PBS+808nm激光1W/cm2照射10分钟)、纳米药物激光不照射组(每孔加入100μL纳米药物,纳米药物浓度分别为0.1、0.2、0.5、1mg/mL)和纳米药物+激光照射组(每孔加入100μL纳米药物,纳米药物浓度分别为0.1、0.2、0.5、1mg/mL,再辅以808nm激光1W/cm2照射10分钟)。
(4)肿瘤细胞光热/铁死亡治疗后,弃掉孔中实验样品溶液,用PBS洗涤3次。
(5)每孔加入195μL染色缓冲液、3μL Hoechst 33342溶液和2μL PI溶液,混匀,置4℃染色30分钟。
(6)将细胞板置荧光显微镜下观察细胞凋亡情况。
(7)CCK-8定量检测抗肿瘤性能实验同上步骤类似。肿瘤细胞经治疗处理后,弃掉细胞板中液体,用PBS洗涤三次。每孔加入100μL CCK-8检测液,酶标仪中450nm处测地吸光度值。
如图4所示,为纳米药物体外光热/铁死亡抗肿瘤治疗后细胞活死双染色结果。从图4中可以看出,随着纳米药物浓度逐渐增高(从0.1mg/mL增加到1mg/mL),激光照射组死亡细胞(红色荧光)逐渐增加并至全部死亡(1mg/mL),而激光未照射组细胞活性不受影响,均存活(蓝色荧光)。该实验证实,在激光照射下,纳米药物可高效的抑制肿瘤细胞的活性,其抗肿瘤能力和浓度呈正相关。而纳米药物本身,在激光未照射时,则表现出良好的生物安全性,对细胞生长无影响。
实施例5
具有光热治疗/铁死亡功能的纳米药物体内抗肿瘤性能检测,步骤如下:
(1)该实施例以乳腺癌4T1为例,但不限于此。
(2)乳腺癌4T1荷瘤小鼠原位瘤瘤模型构建:取20只Balb/c小白鼠,腹部剃毛后,于第四对乳腺部位皮下注射5万个4T1细胞(悬浮于100μL细胞培养基中)。
(3)约10天后,待肿瘤生长至4.5~5mm时,将小鼠随机分为4组(n=5)。
(4)分为4个实验组:PBS组(瘤中注射100μL PBS)、PBS+激光治疗组(瘤中注射100μL PBS+808nm激光1W/cm2照射10分钟)、纳米药物组(瘤中注射100μL纳米药物,10mg/mL)和纳米药物+激光治疗组(瘤中注射100μL纳米药物,10mg/mL+808nm激光1W/cm2照射10分钟)。
(5)每3天拍照记录小鼠肿瘤生长情况,并测量肿瘤大小、监测小鼠体重变化。小鼠肿瘤体积计算公式为:肿瘤体积(mm3)=长(mm)×宽(mm)×宽(mm)/2。
如图5所示,为纳米药物对乳腺癌原位瘤光热/铁死亡协同治疗结果。从图5中可以看出,纳米药物+激光治疗组,荷瘤小鼠肿瘤生长受到抑制,生长缓慢,治疗18天后,肿瘤体积小于50mm3。而三个对照组(纳米药物,PBS+激光照射,PBS)小鼠肿瘤均持续快速生长,其肿瘤大小与“纳米药物+激光照射组”存在统计学显著性差异。该结果表明,我们构建的纳米药物,在近红外激光照射下,可在体内高效的杀死肿瘤细胞,抑制小鼠肿瘤的生长,具备优异的抗肿瘤能力。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案,而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细地说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (10)
1.一种三元硫族金属纳米粒子的制备方法,其特征在于,包括以下步骤:
S1.将铁源试剂、银源试剂和配体混合溶于有机溶剂1中,得到混合物;所述铁源试剂、银源试剂或配体中至少有一种含有硫元素;
S2.在惰性气体氛围中,25~35℃温度下,将步骤S1得到的混合物搅拌15~60min;然后升温至80~95℃,维持10~30min;再升温至150~240℃,反应10~30min;
S3.在步骤S2反应之后的体系中加入有机溶剂2,并以7500~9000rpm的转速离心5~15min,保留沉淀物,纯化,即得。
2.根据权利要求1所述三元硫族金属纳米粒子的制备方法,其特征在于,所述铁源试剂为二乙基二硫代氨基甲酸铁,配体为油酸和油胺。
3.根据权利要求1所述三元硫族金属纳米粒子的制备方法,其特征在于,所述铁源试剂为FeCl2,所述配体为油酸和十二硫醇。
4.根据权利要求1所述三元硫族金属纳米粒子的制备方法,其特征在于,所述银源为AgNO3,AgCl或二乙基二硫代氨基甲酸银。
5.根据权利1所述三元硫族金属纳米粒子的制备方法,其特征在于,所述步骤S1中,有机溶剂1为油胺;所述步骤S3中,有机溶剂2为乙醚和/或丙酮。
6.根据权利要求1所述三元硫族金属纳米粒子的制备方法,其特征在于,所述步骤S3中,纯化的步骤包括:将步骤S3得到的沉淀物重悬于甲苯或氯仿中;过滤之后洗涤,然后分散于正己烷中,然后加入浓度为0.001~0.01M的四氟硼酸亚硝的二氯甲烷溶液,搅拌6~12h,离心收集,即得。
7.权利要求1至6任一项所述方法制备得到的三元硫族金属纳米粒子。
8.权利要求7所述三元硫族金属纳米粒子在制备抗癌药物中的应用。
9.权利要求8所述应用,其特征在于,包括以下步骤:
S11.将所述三元硫族金属纳米粒子加入溶剂中,然后加入亲水化修饰的高分子,室温搅拌12~24h,离心收集产物;
S12.将步骤S11得到产物与肿瘤靶向分子反应,纯化后,即得抗癌药物;
其中,步骤S11中溶剂为N,N-二甲基甲酰胺,二甲基亚砜或乙腈中的一种;所述亲水化修饰的高分子为DSPE-PEG2000或DSPE-PEG2000-R,R为氨基、羧基或巯基。
10.根据权利要求9所述应用,其特征在于,所述肿瘤靶向分子叶酸或cRGD。
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