CN110448699A - 包含功能性多肽修饰七甲川花菁素类染料的肿瘤细胞核靶向载药纳米粒子及制备方法 - Google Patents
包含功能性多肽修饰七甲川花菁素类染料的肿瘤细胞核靶向载药纳米粒子及制备方法 Download PDFInfo
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Abstract
本发明提供一种包含功能性多肽修饰七甲川花菁素类染料的肿瘤细胞核靶向载药纳米粒子及制备方法和应用,对七甲川花青素类染料进行功能性多肽直接修饰的方法,改善七甲川花青素类染料水溶性以及提高其光学稳定性,并将其与蒽环化疗药物共载,获得一种具有肿瘤细胞核靶向功能的载药纳米粒子。本发明实现了七甲川花青素类染料功能性多肽的直接修饰,有效改善七甲川花菁素类染料水溶性差的问题,共载蒽环化疗药物制成的载药纳米粒子,增强了肿瘤细胞对载药纳米粒子的摄取,并实现肿瘤细胞核近核区域聚集分布,实现了对肿瘤细胞核的靶向,提高了肿瘤细胞对光敏剂的摄取。
Description
技术领域
本发明属于医药技术领域,尤其是涉及一种包含功能性多肽修饰七甲川花菁素类染料的肿瘤细胞核靶向载药纳米粒子及制备方法。
背景技术
乳腺癌是严重威胁女性生命健康的恶性肿瘤之一。据最新数据统计,世界范围内乳腺癌发病率与死亡率分别为29%与15%。在中国乳腺癌发病率近年来呈显著上升趋势,已居女性癌症发病的首位。乳腺癌的临床治疗手段包括手术、放疗、化疗和免疫治疗等,但这些方法都有着各自的局限。例如,化学药物治疗易使乳腺癌细胞产生多药耐药,降低药物疗效,并且多数化疗药物缺乏选择性,具有不可忽视的毒副作用。因此,目前临床乳腺癌治疗仍然避免不了30%左右的患者出现后期的转移与复发。由此可见,探索安全有效的治疗方法和策略对临床乳腺癌的治疗具有极其重要的理论意义和应用价值。
光学疗法(phototherapy)是一种先进的、非侵入性的肿瘤治疗手段,主要包括两类,光热治疗(photothermal therapy,PTT)和光动力治疗(photodynamic therapy,PDT)。光热治疗是利用光热转换剂(光敏剂)在远程激发光源的照射下吸收光能并将光转换成热能,进而使肿瘤细胞达到一定温度导致其死亡的一种新兴治疗方法。这种治疗方法具有时间及空间可控、以及对周围正常组织的伤害较低等优势,近年来引起了肿瘤研究领域的广泛关注。光动力疗法是利用特定波长的激光激发光敏剂,与肿瘤部位的氧作用,产生大量的活性氧(ROS,主要包括单线态氧与活性氧自由基),进而通过直接杀伤肿瘤细胞和间接作用于肿瘤微环境(抑制肿瘤血管生成,并激活肿瘤免疫等)来消除肿瘤的治疗方法。与传统方法相比,光学疗法具有以下优势:能够选择性消灭原发与复发肿瘤,避免正常组织的损伤;安全、微创,能缩小手术范围,改善患者愈后;能激活肿瘤免疫,减少复发。
目前报道的光学疗法治疗癌症使用的光热材料都是定位在细胞质,而细胞核是遗传物质核酸产生和发挥作用的场所,主要由核膜包裹着大部分的DNA和蛋白质组成,被誉为细胞的大脑和心脏。因此如果将光热效应或光动力效应作用于细胞核内,便可以直接破坏遗传物质,使其无法发挥正常的功能,从而能够更加快速有效的杀死细胞。也就是说,如果能先破坏细胞核,从细胞核的水平杀死癌细胞,将会大大提高癌细胞的死亡效率,达到彻底杀死癌细胞,消除残余癌细胞生长成肿瘤组织的风险,阻止了癌症的复发,提升癌症的治疗效果。
七甲川花青素类染料IR780是一种极具代表性的疏水近红外荧光探针,广泛应用于体内外荧光成像和光学治疗。然而,其较弱的水溶性,以及在亲水性环境中的光学稳定性较差限制了其在生物学领域的应用。这一类染料缺少活性较高的反应基团,很难与高分子直接发生化学反应,通常是在合成过程中在其两端的侧链引入-COOH,然后再和高分子进行偶联,如IR825和IR820。有文献报道,其处于中心位置的氯原子可以和巯基发生亲核取代反应,直接进行PEG化修饰,大大改善了其水溶性,并同时提高了其光学稳定性,提高了对肿瘤的光热治疗效果。但是,利用多肽对IR780直接进行修饰尚未见报道。
近年来,越来越多的研究表明利用多功能纳米载体能够将PDT与传统的化疗结合,具有显著的抗肿瘤疗效。除此以外,多功能纳米载体还为药物的靶向递送与可控释放提供了可能,不仅可以实现抗肿瘤协同增效作用,还能有效减少药物的毒副作用。
发明内容
为解决上述技术问题,本发明提供一种包含功能性多肽修饰七甲川花菁素类染料的肿瘤细胞核靶向载药纳米粒子及制备方法。
本发明采用的技术方案是:一种功能性多肽直接修饰的七甲川花菁素类染料,在碱性条件下,以功能性多肽的巯基亲核取代七甲川花青素类染料的氯原子;
优选地,七甲川花菁素类染料为IR780,IR808,IR825或IR-DBI。
优选地,功能性多肽包括细胞穿膜肽,血管靶向肽,细胞穿透肽或淋巴结靶向肽;
优选地,细胞穿膜肽为TAT,R9或LMWP;
优选地,血管靶向肽为RGD,cRGD或iRGD;
优选地,细胞穿透肽为NGR;
优选地,淋巴结靶向肽为Lyp-1。
功能性多肽直接修饰的七甲川花菁素类染料的合成方法,将功能性多肽,七甲川花菁素类染料和三乙胺溶于溶剂中形成反应体系,以25-60℃条件避光搅拌 24-96h,将反应后的溶液置于蒸馏水中透析12-48h,冷冻干燥后获得功能性多肽直接修饰的七甲川花菁素类染料;
优选地,溶剂为二甲基亚砜;
优选地,功能性多肽,七甲川花菁素类染料和三乙胺摩尔比为1.2:1:1.8;
优选地,透析截留分子量为500D;
优选地,反应体系预先通氮2min,40℃条件避光搅拌72h;
优选地,透析24h。
一种具有肿瘤细胞核靶向的多功能载药纳米粒子,以功能性多肽直接修饰的七甲川花菁素类染料为两亲性材料共载蒽环化疗药物获得的载药纳米粒子,其结构为核壳结构,功能性多肽为穿膜肽。
优选地,七甲川花菁素类染料与蒽环类化疗药物通过π-π共轭作用形成疏水内核。
优选地,蒽环化疗药物包括阿霉素、柔红霉素、表阿霉素、伊达比星中的一种或多种。
具有肿瘤细胞核靶向的多功能载药纳米粒子的制备方法,将蒽环化疗药物进行脱盐处理,制备蒽环化疗药物DMSO溶液,制备功能性多肽直接修饰的七甲川花菁素类染料DMSO溶液,二者混合后避光搅拌,滴加到等体积超纯水中搅拌获得载药纳米粒子原溶液,再取小剂量载药纳米粒子原溶液滴入到超纯水中搅拌获得载药纳米粒子溶液;
优选地,蒽环化疗药物DMSO溶液终浓度为10mg/mL;
优选地,功能性多肽直接修饰的七甲川花菁素类染料DMSO溶液终浓度为 50mg/mL;
优选地,以七甲川花菁素类染料与蒽环化疗药物质量比为2:1的比例进行混合;
优选地,搅拌15-20min获得载药纳米粒子原溶液;
优选地,取39μL载药纳米粒子原溶液滴入到361μL的DDW中,搅拌 30-40min获得载药纳米粒子溶液。
功能性多肽直接修饰的七甲川花菁素类染料在对七甲川花菁素类染料进行高分子修饰中的应用。
具有肿瘤细胞核靶向的多功能载药纳米粒子在制备肿瘤细胞核靶向递送药物中的应用;
优选地,肿瘤细胞核靶向递送药物为细胞核靶向的光热/光动力与化疗协同的多功能抗肿瘤肿瘤纳米药物。
本发明具有的优点和积极效果是:
1通过功能性多肽对七甲川花菁素类染料进行修饰,能够有效改善七甲川花菁素类染料水溶性差的问题,还能够为七甲川花菁素类染料提供多肽直接修饰的靶点;并能够大幅提高七甲川花菁素类染料的光学稳定性;
2将功能性多肽直接修饰的七甲川花菁素类染料共载蒽环化疗药物获得的纳米粒子,其结构更加致密,能够显著提高七甲川花菁素类染光热性能,并且其制备方法简单,成本低廉,适于大规模生产的需求;
3功能性多肽,例如穿膜肽显著增强了肿瘤细胞对载药纳米粒子的摄取,并实现肿瘤细胞核近核区域聚集分布,实现了对肿瘤细胞核的靶向;制备获得的载药纳米体系能够实现肿瘤细胞核靶向的光热、光动力与化疗的联合治疗,具有显著的协同增效作用,在体内外表现出较强抗肿瘤疗效。
附图说明
图1是实施例1中TAT-IR780偶联物的合成路线、TID纳米粒子制备以及抗肿瘤作用机制示意图;
图2是实施例1中IR780,TAT以及反应后得到的偶联物TAT-IR780的红外光谱图;
图3是实施例1中IR780,TAT以及反应后得到的偶联物TAT-IR780的核磁 (1H NMR)图谱;
图4是实施例1中TAT(左)以及TAT与IR780反应后得到的偶联物 TAT-IR780(右)的质谱图;
图5是实施例1中TAT-IR780组装的纳米粒子TIR的透射电镜照片;
图6是实施例1中TAT-IR780组装的纳米粒子TIR的粒径分布图;
图7是实施例1中共载蒽环类化疗药物阿霉素(DOX)的纳米粒子TID的透射电镜照片;
图8是实施例1中共载蒽环类化疗药物阿霉素(DOX)的纳米粒子TID的粒径分布图;
图9是实施例1中PBS,DOX,IR780,TIR与TID溶液在激光照射下的升温曲线图;
图10是实施例1中TID纳米粒子溶液在不同pH的PBS以及结合激光照射下体外药物释放曲线图;
图11是实施例1中小鼠乳腺癌4T1细胞对游离药物DOX,IR780和TIR,TID 纳米粒子的摄取以及细胞内的分布情况;
图12是实施例1中游离药物DOX,IR780和TIR,TID纳米粒子在小鼠乳腺癌4T1细胞3D细胞球中的渗透与分布情况;
图13是实施例1中游离药物DOX,IR780和TIR,TID纳米粒子在有无激光照射下对4T1细胞的毒性作用;
图14是实施例1中TID纳米粒子结合激光照射处理后4T1细胞内热休克蛋白60(HSP60)的表达;
图15是实施例1中TID纳米粒子结合激光照射处理后4T1细胞核内DNA 双链损伤相关蛋白γ-H2AX表达和53BP1分布状态观察;
图16是实施例1中用活性氧探针DCFH-DA检测到的载药纳米粒子TID与 4T1细胞共孵育后在有无激光照射下的活性氧分布情况;
图17是实施例1中荷瘤小鼠瘤内给予载药纳米粒子TID后在激光照射下肿瘤的升温曲线;
图18是实施例1中载药纳米粒子TID结合激光照射对4T1荷瘤小鼠体内肿瘤生长的抑制曲线;
图19是实施例1中载药纳米粒子TID结合激光照射对4T1荷瘤小鼠治疗结束后的肿瘤照片。
具体实施方式
本发明旨在提供一种七甲川花青素类染料直接进行功能性多肽修饰的方法,以及包含这种功能性多肽修饰七甲川花菁素类染料的能够靶向肿瘤细胞核的纳米粒子的制备方法与应用。该载药纳米粒子不仅可以实现七甲川花青素光敏分子与蒽环类化疗药物的共载,还可以通过功能性多肽,例如穿膜肽的穿膜与细胞核靶向作用,提高肿瘤细胞对光敏剂摄取,并将药物高效递送至肿瘤细胞近核区域,随后激光照射激活七甲川花青素类染料的光热效应,产生高热,更近距离地对肿瘤细胞核遗传物质产生热损伤,诱导肿瘤的急性坏死、细胞凋亡以及微环境破坏等;同时激光照射过程中,光敏剂还伴随一定的光动力效应,产生大量活性氧自由基,损伤肿瘤细胞线粒体,破坏细胞内呼吸平衡,激活线粒体介导的细胞凋亡通路,进而诱导细胞凋亡;另外高热能够进一步促进化疗药物的释放,发挥化疗作用,以此实现光热治疗(PTT)、光动力治疗(PDT)与化疗多种疗法的有效联合,进而发挥协同增效的抗肿瘤作用。
功能性多肽直接修饰的七甲川花菁素类染料,以多肽的巯基与七甲川花青素类染料的氯原子发生亲核取代反应为原理,在不影响多肽功能的前提下在序列末端连接半胱氨酸,使其具有巯基基团,所述巯基与氯原子的取代反应在碱性条件下进行,并脱去一分子氯化氢。其制备方法是将功能性多肽,七甲川花菁素类染料和三乙胺溶于二甲基亚砜中形成反应体系,功能性多肽,七甲川花菁素类染料和三乙胺摩尔比为1.2:1:1.8,反应体系预先通氮2min,以25-60℃条件避光搅拌 24-96h,将反应后的溶液置于蒸馏水中透析12-48h,透析截留分子量为500D,冷冻干燥后获得功能性多肽直接修饰的七甲川花菁素类染料。
再以功能性多肽直接修饰的七甲川花菁素类染料为两亲性材料共载蒽环化疗药物获得的具有肿瘤细胞核靶向的多功能载药纳米粒子,七甲川花菁素类染料与蒽环类化疗药物通过π-π共轭作用形成疏水内核,形成核壳结构。其制备方法是将蒽环化疗药物溶于甲醇中,按照3当量加入TEA搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为10mg/mL;功能性多肽直接修饰的七甲川花菁素类染料溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照七甲川花菁素类染料与蒽环化疗药物质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL的DDW中,搅拌30min即得载药纳米粒子溶液。
例如,该具有肿瘤细胞核靶向功能的载药纳米粒子以具有细胞核靶向功能的细胞穿膜肽TAT为功能性多肽,以IR780为七甲花青素类染料的代表,利用其末端的半胱氨酸巯基与IR780的氯原子发生取代反应合成TAT-IR780,并利用 IR780与蒽环类化疗药物DOX之间的π-π共轭以及疏水相互作用形成具有疏水性内核的纳米粒子。TAT大大提高肿瘤细胞对IR780的摄取,并高效递送至近核区域,在近红外激光的照射下,激发光敏剂光热和光动力效应,发挥细胞核靶向的光热和光动力作用,携载的化疗药物阿霉素进入细胞核发挥化疗作用。
该具备肿瘤细胞核靶向功能的载药纳米粒子的制备方法:
1)TIR偶联物的合成。
将TAT(120mg,72μmol),IR780(40mg,60μmol)和三乙胺(15μL,108μmol) 溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR780。
2)具有肿瘤细胞核靶向功能的载药纳米粒子TID的制备。
①将盐酸阿霉素(DOX·HCl)溶于甲醇中,浓度为2mg/mL,按照3当量加入三乙胺(TEA)搅拌6h,进行脱盐处理,随后旋蒸除去甲醇,加入DMSO 复溶,使终浓度为10mg/mL。
②TAT-IR780溶于DMSO,浓度为50mg/mL,与脱盐后的DOX按照 IR780:DOX=2:1的质量比混合,避光下搅拌10min,随后将该溶液滴加入等体积的超纯水(DDW)中,避光下搅拌15min后,然后将该体系缓慢滴加于9倍量的DDW中,再避光搅拌30min即得载药纳米粒子TID溶液。同时以相同方法制备不含DOX的非载药纳米粒子TIR。最终制备得到的纳米溶液中IR780浓度为400μg/mL,DOX浓度为200μg/mL
经分析,制备所得的载药纳米粒子水溶性较好,粒径约110nm左右,电位约+30mV,在DDW中的稳定性良好,电镜下观察具有致密的球形结构,分布均匀;紫外与荧光稳定性考察发现IR780与TAT偶联之后,紫外与荧光的稳定性大大增强;体外红外热成像分析可知,与游离的IR780和TIR相比,TID具有良好的光热效应,且在随后的体外释放实验中,光照及pH=6.5的条件下TID表现出更多的DOX释放;细胞实验显示与游离药物及TIR相比,载药纳米粒子TID 能够被乳腺癌4T1细胞更多的摄取,同时在激光照射后表现出比游离药物和不施加光照的载药纳米粒子组更强的细胞毒性;体内药效学实验显示,该纳米载药粒子在肿瘤组织中仍能将光敏剂带至细胞近核区域,并在激光照射作用下较大程度上消融肿瘤,并对后期肿瘤的复发具有较强的抑制作用。
本方案中,七甲川花菁素类染料还可以为IR808,IR825或IR-DBI;功能性多肽可以为细胞穿膜肽,血管靶向肽,细胞穿透肽或淋巴结靶向肽;细胞穿膜肽可选用TAT,R9或LMWP,血管靶向肽可选用RGD,cRGD或iRGD,细胞穿透肽可选用NGR,淋巴结靶向肽可选用Lyp-1;蒽环化疗药物可为阿霉素、柔红霉素、表阿霉素、伊达比星中的一种或多种化疗药物的组合。下面以细胞穿膜肽 TAT修饰IR780,再与阿霉素DOX共载形成载药纳米例子为代表,对本方案进行具体描述,它们只用于对本发明进行进一步的说明,不能理解为对本发明保护范围的限制,本领域的技术人员根据上述本发明的内容做出一些非本质的改进和调整,均属本发明保护范围。
实施例1:
1、TAT-IR780的合成与表征
将TAT(120mg,72μmol),IR780(40mg,60μmol)和三乙胺(15μL,108μmol) 溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR780。合成路线如图1所示,TAT-IR780的化学结构通过IR图谱(图2)、1H NMR谱(图3)和质谱(图4)进行表征。最终合成得到的TAT-IR780在IR图谱和1H NMR谱中同时具有IR780和TAT的特征峰,根据质谱分子离子峰,按以下公式计算分子量得TAT分子量为1662,TAT-IR780 分子量为2165,与产物理论上分子量一致,以上充分证明TAT-IR780成功合成。
2、TAT-IR780高效负载蒽环类化疗药物DOX的载药纳米粒子TID的制备与表征
将蒽环化疗药物盐酸阿霉素(DOX·HCl)溶于甲醇中,按照3当量加入TEA 搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为10 mg/mL;TAT-IR780溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR780与DOX质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL 的DDW中,搅拌30min即得TID载药纳米粒子溶液。以空白DMSO替代DOX,按上述相同方法制备得到TIR非载药纳米粒子。
在超纯水中,非载药纳米粒子TIR的透射电镜照片和粒径分布情况如图5 和图6所示,可以观察到其粒径约为100nm左右,分布相对均匀,结构相对致密,电位为35.6mV;载药纳米粒子TID的透射电镜照片和粒径分布情况如图7 和图8所示,可以观察到其粒径约为110nm左右,形貌较为规则球形,并具有一定的核壳结构,分布较为均匀,结构较为致密,电位为32.4mV。
3、载药纳米粒子TID的光热性能考察
随后对载药纳米粒子TID水溶液的体外光热效应进行了考察,结果如图5 所示。将1mL各不同溶液(含IR780浓度为50μg/mL,DOX浓度为25μg/mL) 置于5mL离心管中,然后使用785nm近红外激光对其进行照射,激光照射过程中使用红外热成像照相机记录光照过程中的时间变化,以照射时间为横坐标,温度为纵坐标作图。从图中可以看出,TAT偶联IR780后对IR780的光热效应无明显影响,但载药纳米粒子的光热效应大大增强,原因可能是TIR中的IR780 与DOX之间具有π-π共轭作用和疏水相互作用,使其结构更加致密,对近红外激光的吸收能力变强。
4、载药纳米粒子TID的光热响应性释药性能考察
取四份1.5mL载药纳米粒子TID的分散液,对其中两份施加强度为1W/cm2的激光照射,持续照射10min,随后将其置于截留分子量为1000的透析袋中,以光照与不光照为一组,将装载两组溶液的透析袋分别置于20mL pH=6.5和 pH=7.4的PBS释放介质中,在37℃恒温摇床中避光震荡(100rpm)。在规定时间点1,2,4,8,12,24h,48h,60h及72h取释放介质2mL,并加入等体积的新鲜 PBS。利用荧光分光光度计测定释放液在激发波长为480nm,发射波长为570nm 的荧光值,代入建好的游离DOX荧光值与浓度关系的标准曲线中,计算释放液中DOX的浓度,DOX释放百分率按照以下公式计算:
释放百分率%=(药物释放量/药物总含量)×100%
载药纳米粒子TID在有无激光照射和不同pH条件下的释药曲线如图10所示,DOX的释放在激光照射后显著加快,且在pH=6.5的条件下释放量更高,提示TID能够响应激光释放DOX,且在弱酸性的肿瘤微环境中更容易发挥药效。
5、小鼠乳腺癌4T1细胞对载药纳米粒子TID的摄取能力及其亚细胞分布检测
将置于DMEM FULL细胞培养液中的4T1细胞常规消化、离心、重悬、计数,随后分别以5×104个/孔的密度接种于玻璃底12孔板之中,放入37℃,5% CO2培养箱中孵育24h,弃去培养基,分别加入1mL经DMEM FULL培养液稀释过的DOX、IR780、TIR以及载药纳米粒子TID,DOX和IR780的浓度分别为2μg/mL和4μg/mL。继续孵育6h后,弃去培养液,用PBS洗2次,4%多聚甲醛固定10min,再用以1:900稀释于PBS中的DAPI染色10min,最后于激光共聚焦显微镜下观察,通过DOX与IR780的自发荧光判断细胞对不同药物的摄取能力,结果如图11所示。结果表明,经TAT修饰后,4T1细胞对IR780的摄取大大增强,并且集中分布于细胞核附近,具有较好的细胞核趋向作用。
6、载药纳米粒子TID在4T1细胞3D细胞球中的渗透性
以DMEM培养液为溶剂在高温下配制浓度为1.5%(w/v)的无菌琼脂糖溶液,以60μL/孔对玻璃底的96孔板进行包被,然后置于4℃过夜。将培养中的 4T1细胞经消化、离心、重悬制成浓度为5×104个/mL的细胞悬液,然后加入一定体积的基质胶(终浓度为2.5%v/v)。接着以200μL/孔接种于包被的96孔板中,4℃条件下,1000g离心10min,于细胞培养箱中继续培养,每2天取出100 μL原培养基换成100μL新鲜培养基,待肿瘤细胞球长到400μm,弃去100μL 原培养基,换成100μL含游离药物和纳米粒子的新鲜培养基(IR780浓度为20 μg/mL,DOX浓度为10μg/mL)。孵育6h后于激光共聚焦下观察IR780和DOX 荧光在肿瘤细胞球中的分布,结果如图12所示。从图中可以看出游离IR780较多分布于细胞球的外围边缘区域,而TIR和TID较多分布于细胞球中心区域,表明经TAT修饰后,IR780在肿瘤细胞球中的渗透作用大大增强。
7、载药纳米粒子TID结合激光照射对4T1细胞的毒性作用
将对数生长期的4T1细胞以6×103个/孔的密度接种于96孔板之中,放入 37℃,5%CO2培养箱中孵育24h,吸去培养基,按照以下分组给药:空白对照、 DOX±激光照射、IR780±激光照射、TIR±激光照射以及TID±激光照射。药物与细胞共孵育6h后,对相应组别的细胞施加785nm、强度为1W/cm2的激光照射,持续照射5min,随后转入孵箱中培养18h,弃液,向每孔加入120μL(0.83mg/mL) MTT溶液,继续放入孵箱中培养3h,弃去培养液,每孔加入150μL DMSO,并置于摇床上以100rpm的转速震荡5min,最后用酶标仪检测样品在490nm处的光密度吸光度值即OD值。设置不加药组为空白对照,按照以下公式计算细胞存活率:
存活率=(实验孔OD值/对照孔OD值)×100%
4T1细胞经过不同处理后的细胞活性变化如图13所示,相比于单独的光动力治疗组或单独化疗组,载药纳米粒子TID联合激光照射治疗表现出更显著的细胞生长抑制效果,IC50值明显降低,表明TID联合激光照射治疗具有更好地杀伤肿瘤细胞的作用。为评价该载药纳米胶束体系联合两种药物的协同抗肿瘤效果,利用以下公式计算协同指数(CI):
CI=IC50(TID+激光照射组中IR浓度)/IC50(IR780+激光照射组中IR780浓度)+IC50(TID+激光照射组中DOX浓度)/IC50(TID组中DOX浓度)
计算结果为CI=0.899,根据标准(CI>1为拮抗作用,CI=1为叠加作用,CI<1 为协同作用)判断,载药纳米粒子TID结合激光照射后表现出显著的协同抗肿瘤作用,实现了PTT,PDT与化疗的有效联合。
8、载药纳米粒子TID结合激光照射诱导4T1细胞热休克蛋白60表达
首先,在12孔板底部铺上玻璃片,取对数生长的4T1细胞,以5×104个/孔接种于玻片上,放入37℃,5%CO2培养箱中孵育24h,吸去培养基,换成含药培养基,按照以下分组给药:空白对照、DOX、IR780±激光照射、TIR±激光照射、TID±激光照射,其中IR780和DOX的浓度分别为1μg/mL和0.5μg/mL。药物与细胞共孵育6h后,对相应组别的细胞施加785nm、强度为1W/cm2的激光照射,持续照射5min,随后转入孵箱中继续培养12h。最后洗去药液,细胞经4%多聚甲醛固定,0.2%Triton X-100打孔,抗HSP60一抗染过夜、荧光素647 标记的二抗染色以及DAPI染细胞核后,将细胞置于激光共聚焦下观察,结果如图14所示,相比于游离IR780,TIR和TID联合激光照射能够刺激4T1细胞更多的热休克蛋白,证明TIR和TID联合激光照射对4T1细胞具有较好地光热治疗效果。
9、载药纳米粒子TID结合激光照射诱导4T1细胞热休克蛋白60表达
将处于对数生长期的4T1细胞以5×104个/孔接种于12孔板中的玻片上,放入37℃,5%CO2培养箱中孵育24h,吸去培养基,换成含药培养基,按照以下分组给药:空白对照、DOX、IR780±激光照射、TIR±激光照射、TID±激光照射,其中IR780和DOX的浓度分别为1μg/mL和0.5μg/mL。药物与细胞共孵育6h 后,对相应组别的细胞施加785nm、强度为1W/cm2的激光照射,持续照射5min,随后转入孵箱中继续培养12h。最后洗去药液,细胞经4%多聚甲醛固定,0.2% Triton X-100打孔,抗γ-H2AX和抗53BP1一抗共染过夜、荧光素647标记的二抗和FITC标记的二抗染色以及DAPI染细胞核后,将细胞置于激光共聚焦下观察,结果如图15所示。结果表明,载药纳米粒子TID结合激光照射能够能够明显上调H2AX蛋白磷酸化水平,并同时较大程度上引起53BP1蛋白的聚集,证明接受TID结合激光照射处理后,肿瘤细胞核双链DNA受到明显的损伤,表明 TID具有细胞核靶向的光热光动力与化疗联合的抗肿瘤作用。
10、载药纳米粒子TID结合激光照射后细胞内活性氧产量检测
将处于对数生长期的4T1细胞以5×104个/孔接种于12孔板中的玻片上,放入37℃,5%CO2培养箱中孵育24h,吸去培养基,换成含药培养基,按照以下分组给药:空白对照、DOX、IR780±激光照射、TIR±激光照射、TID±激光照射,其中IR780和DOX的浓度分别为1μg/mL和0.5μg/mL。药物与细胞共孵育6h 后,对相应组别的细胞施加785nm、强度为1W/cm2的激光照射,持续照射2min,随后转入孵箱中继续培养12h,弃去培养液后用PBS液洗3次,经过DCFH-DA 染料染色、4%多聚甲醛固定以及DAPI染细胞核之后,将细胞置于激光共聚焦显微镜下观察胞内绿色荧光信号,其强度与活性氧产量成正相关。如图16所示,相比于DOX、IR780、TIR治疗组,IR780+激光照射、TIR+激光照射、TID+激光照射组细胞内生成了大量的活性氧自由基,且TIR和TID联合激光照射组活性氧产量比游离IR780联合激光照射组明显升高。以上数据说明载药纳米粒子 TID结合激光照射产热的过程中还能够最大程度地提升4T1细胞内活性氧水平,具有较强的光动力效应。
11、载药纳米粒子TID体内光热效应考察
首先建立小鼠乳腺癌4T1移植瘤模型的:将处于对数生长期的4T1细胞常规消化,离心,收集,再用PBS清洗2遍,以彻底除去培养液中的血清。随后进行细胞计数,用无菌PBS调整细胞浓度至4×106个/mL,用注射器抽取细胞悬液,向每只裸鼠的右肩部皮下注射100μL。待移植瘤直径生长到约为8mm时,开始进行体内光热效应考察。将100μL PBS,IR780,TIR和TID溶液直接打到肿瘤部位,其中IR780浓度为400μg/mL,DOX浓度为200μg/mL。6h后对小鼠肿瘤部位给与波长为785nm,功率为1.0W/cm2的近红外激光照射,时间为5min,期间用红外热成像照相机记录升温过程,每15s拍摄一张照片,以照射时间为横轴,温度为纵轴做升温曲线图,结果如图17,所示。在体内,TID比TIR和 IR780的光热效应稍好一些,原因可能是IR780在体内结合了蛋白,进入其疏水结构与,对近红外激光的吸收能力变强。总之,载药纳米粒子结合激光照射在体内也表现出较好的光热效应。
12、载药纳米粒子TID结合激光照射的体内抗肿瘤作用评价
待荷瘤小鼠肿瘤长至100mm3,将其随机分为8组,每组6只,按照以下方式进行治疗:PBS对照组;游离DOX组;游离IR780组;游离IR780+激光照射组;TIR组;TIR+激光照射组;TID组;TID+激光照射组,以上分组中涉及到的IR780剂量均为1mg/kg,DOX的剂量均为0.5mg/kg。经瘤内给药后6h,联合激光照射组给与波长为785nm,功率为1.0W/cm2的近红外激光照射,持续时间为5min,之后各组小鼠正常饲养。期间,每两天测量一次肿瘤的长(a)和宽 (b),并按照公式V=ab2/2计算即得,绘制肿瘤生长曲线。治疗结束后,将各组小鼠肿瘤解剖下来,进行拍照。
如图18,图19所示,IR780与TAT偶联后,在激光照射下表现出较好的肿瘤消融作用,载药纳米粒子TID结合激光照射能够较大程度上消融肿瘤,并对后期肿瘤的复发同时具有较强的抑制作用,说明该纳米载药体系实现的细胞核靶向的光热/光动力与化疗对乳腺癌表现出较好的协同作用。
实施例2:
1、穿膜肽R9偶联IR780的合成
将R9(序列RRRRRRRRRC)多肽(110mg,72μmol),IR780(40mg,60μmol) 和三乙胺(15μL,108μmol)溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的R9多肽,冷冻干燥后即得深绿色粉末状偶联物R9-IR780。
2、R9-IR780高效负载蒽环类化疗药物DOX的载药纳米粒子RID的制备与表征
将蒽环化疗药物盐酸阿霉素(DOX·HCl)溶于甲醇中,按照3当量加入TEA 搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为10 mg/mL;R9-IR780溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR780与DOX质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL的 DDW中,搅拌30min即得RID载药纳米粒子溶液。以空白DMSO替代DOX,按上述相同方法制备得到RIR非载药纳米粒子。
实施例3:
1、TAT-IR780合成
将TAT(120mg,72μmol),IR780(40mg,60μmol)和三乙胺(15μL,108μmol) 溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR780。
2、TAT-IR780高效负载蒽环类化疗药物表阿霉素(EPB)的载药纳米粒子 TIE的制备
将蒽环化疗药物盐酸表阿霉素(EPB·HCl)溶于甲醇中,按照3当量加入 TEA搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为 10mg/mL;TAT-IR780溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR780与EPB质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL 的DDW中,搅拌30min即得TIE载药纳米粒子溶液。以空白DMSO替代EPB,按上述相同方法制备得到TIR非载药纳米粒子。
实施例4:
1、TAT-IR780合成
将TAT(120mg,72μmol),IR780(40mg,60μmol)和三乙胺(15μL,108μmol) 溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR780。
2、TAT-IR780高效负载蒽环类化疗药物柔红霉素(DNR)的载药纳米粒子 TID的制备
将蒽环化疗药物盐酸柔红霉素(DNR·HCl)溶于甲醇中,按照3当量加入 TEA搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为 10mg/mL;TAT-IR780溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR780与DNR质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL 的DDW中,搅拌30min即得TID载药纳米粒子溶液。以空白DMSO替代DNR,按上述相同方法制备得到TIR非载药纳米粒子。
实施例5:
1、TAT-IR808的合成
将TAT(120mg,72μmol),IR808(41mg,60μmol)和三乙胺(15μL,108μmol) 溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR808。
2、TAT-IR808高效负载蒽环类化疗药物DOX的载药纳米粒子TID的制备与表征
将蒽环化疗药物盐酸阿霉素(DOX·HCl)溶于甲醇中,按照3当量加入TEA 搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为10 mg/mL;TAT-IR808溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR808与DOX质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL 的DDW中,搅拌30min即得TID载药纳米粒子溶液。以空白DMSO替代DOX,按上述相同方法制备得到TIR非载药纳米粒子。
实施例6:
1、TAT-IR825的合成
将TAT(120mg,72μmol),IR825(49.5mg,60μmol)和三乙胺(15μL,108 μmol)溶于12mL二甲基亚砜(DMSO),反应体系通氮气气流,避光条件下40℃搅拌反应72h,随后将反应液转移至透析袋(截留分子量为500Da)中,并于超纯水中透析24h,每6h更换外透析液,除去未反应的TAT多肽,冷冻干燥后即得深绿色粉末状偶联物TAT-IR825。
2、TAT-IR825高效负载蒽环类化疗药物DOX的载药纳米粒子TID的制备与表征
将蒽环化疗药物盐酸阿霉素(DOX·HCl)溶于甲醇中,按照3当量加入TEA 搅拌8h,进行脱盐处理,随后旋蒸除去甲醇,再用DMSO复溶,终浓度为10 mg/mL;TAT-IR825溶于DMSO,终浓度为50mg/mL,与脱盐后的蒽环化疗药物按照IR825与DOX质量比为2:1进行混合,避光条件下搅拌10min,随后将溶液滴加入等体积的超纯水(DDW)中,搅拌15min后,取39μL滴入到361μL 的DDW中,搅拌30min即得TID载药纳米粒子溶液。以空白DMSO替代DOX,按上述相同方法制备得到TIR非载药纳米粒子。
实施例2-6中的多肽-七甲花青素染料偶联物的合成表征、纳米载药颗粒的形貌与粒径、体外光热效应、体外药物释放、肿瘤细胞摄取能力、细胞球渗透性能、细胞水平抗肿瘤疗效、热休克蛋白表达、DNA损伤、活性氧产量、体内肿瘤治疗效果与实施例1中数据相仿,在此不重复叙述。
以上对本发明的几个实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围。凡依本发明申请范围所作的均等变化与改进等,均应仍归属于本发明的专利涵盖范围之内。
Claims (10)
1.一种功能性多肽直接修饰的七甲川花菁素类染料,其特征在于:在碱性条件下,以功能性多肽的巯基亲核取代七甲川花青素类染料的氯原子。
2.根据权利要求1所述的功能性多肽直接修饰的七甲川花菁素类染料,其特征在于:所述七甲川花菁素类染料为IR780,IR808,IR825或IR-DBI。
3.根据权利要求1所述的功能性多肽直接修饰的七甲川花菁素类染料,其特征在于:所述功能性多肽包括细胞穿膜肽,血管靶向肽,细胞穿透肽或淋巴结靶向肽;
优选地,细胞穿膜肽为TAT,R9或LMWP;
优选地,血管靶向肽为RGD,cRGD或iRGD;
优选地,细胞穿透肽为NGR;
优选地,淋巴结靶向肽为Lyp-1。
4.权利要求1-3中任一所述的功能性多肽直接修饰的七甲川花菁素类染料的合成方法,其特征在于:将功能性多肽,七甲川花菁素类染料和三乙胺溶于溶剂中形成反应体系,以25-60℃条件避光搅拌24-96h,将反应后的溶液置于蒸馏水中透析12-48h,冷冻干燥后获得功能性多肽直接修饰的七甲川花菁素类染料;
优选地,溶剂为二甲基亚砜;
优选地,功能性多肽,七甲川花菁素类染料和三乙胺摩尔比为1.2:1:1.8;
优选地,透析截留分子量为500D;
优选地,反应体系预先通氮2min,40℃条件避光搅拌72h;
优选地,透析24h。
5.一种具有肿瘤细胞核靶向的多功能载药纳米粒子,其特征在于:以权利要求1-3中任一所述的功能性多肽直接修饰的七甲川花菁素类染料为两亲性材料共载蒽环化疗药物获得的载药纳米粒子,其结构为核壳结构。
6.根据权利要求5所述的具有肿瘤细胞核靶向的多功能载药纳米粒子,其特征在于:七甲川花菁素类染料与蒽环类化疗药物通过π-π共轭作用形成疏水内核。
7.根据权利要求5所述的具有肿瘤细胞核靶向的多功能载药纳米粒子,其特征在于:所述蒽环化疗药物包括阿霉素、柔红霉素、表阿霉素、伊达比星中的一种或多种。
8.权利要求5-7中任一所述的具有肿瘤细胞核靶向的多功能载药纳米粒子的制备方法,其特征在于:将蒽环化疗药物进行脱盐处理,制备蒽环化疗药物DMSO溶液,制备功能性多肽直接修饰的七甲川花菁素类染料DMSO溶液,二者混合后避光搅拌,滴加到等体积超纯水中搅拌获得载药纳米粒子原溶液,再取小剂量载药纳米粒子原溶液滴入到超纯水中搅拌获得载药纳米粒子溶液;
优选地,蒽环化疗药物DMSO溶液终浓度为10mg/mL;
优选地,功能性多肽直接修饰的七甲川花菁素类染料DMSO溶液终浓度为50mg/mL;
优选地,以七甲川花菁素类染料与蒽环化疗药物质量比为2:1的比例进行混合;
优选地,搅拌15-20min获得载药纳米粒子原溶液;
优选地,取39μL载药纳米粒子原溶液滴入到361μL的DDW中,搅拌30-40min获得载药纳米粒子溶液。
9.权利要求1-3中任一所述的功能性多肽直接修饰的七甲川花菁素类染料在对七甲川花菁素类染料进行高分子修饰中的应用。
10.权利要求5-7中任一所述的具有肿瘤细胞核靶向的多功能载药纳米粒子在制备肿瘤细胞核靶向递送药物中的应用;
优选地,肿瘤细胞核靶向递送药物为细胞核靶向的光热/光动力与化疗协同的多功能抗肿瘤纳米药物。
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