CN114805372B - 具有双摄取通路的轴向磺酸基修饰酞菁硅及其制备方法及和应用 - Google Patents
具有双摄取通路的轴向磺酸基修饰酞菁硅及其制备方法及和应用 Download PDFInfo
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- CN114805372B CN114805372B CN202210395012.3A CN202210395012A CN114805372B CN 114805372 B CN114805372 B CN 114805372B CN 202210395012 A CN202210395012 A CN 202210395012A CN 114805372 B CN114805372 B CN 114805372B
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- JACPFCQFVIAGDN-UHFFFAOYSA-M sipc iv Chemical compound [OH-].[Si+4].CN(C)CCC[Si](C)(C)[O-].C=1C=CC=C(C(N=C2[N-]C(C3=CC=CC=C32)=N2)=N3)C=1C3=CC([C]1C=CC=CC1=1)=NC=1N=C1[C]3C=CC=CC3=C2[N-]1 JACPFCQFVIAGDN-UHFFFAOYSA-M 0.000 claims abstract description 31
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
本发明公开了一种具有双摄取通路的轴向磺酸基修饰酞菁硅及其制备方法和应用。本发明设计的酞菁硅可以通过磺酸阴离子和白蛋白介导的双摄取途径被HepG2细胞吸收,产生极高的光细胞毒性,IC50值为2.0 nM。另外,本发明所述的磺酸基修饰酞菁与白蛋白复合物不仅提高了酞菁的光活性(如荧光发射和活性氧的产生),而且能在血液中的FBS的存在下完全解聚,产生最大的光敏活性。磺酸基修饰酞菁与白蛋白复合物的肿瘤靶向性也显著增强,能够实现有效的体内荧光成像和光动力治疗。这是首个阴离子酞菁通过有机阴离子转运多肽(OATP)途径摄取的例子,也是光敏剂通过分子和超分子介导的双重细胞摄取途径的首个例子。
Description
技术领域
本发明属于光敏剂、光动力治疗药物领域,具体涉及一种具有双摄取通路的轴向磺酸基修饰酞菁硅及其制备方法和应用。
背景技术
侵袭性肿瘤已成为影响人类健康、威胁人类生命的主要疾病之一,但是传统的肿瘤治疗方法包括手术和化疗等均存在较大缺点和不足,致使肿瘤的防治已经成当前急需解决的问题。光动力疗法(Photodynamic therapy, PDT)作为一种全新的治疗手段,因其具有非侵入性、毒副作用小、收效快、重复应用下不会产生耐药性等优点,已成为一种很有前途的癌症治疗方法。PDT是一种基于光化学的治疗方法,该方法依赖于光敏剂(PS)、光和氧三种成分对组织和细胞造成的损伤。在三种成分中,PS是PDT过程的核心物质。因此,PSs的性能对PDT的效率至关重要,被认为是该领域的研究热点。
酞菁(Pc)是一种染料类的PS。作为第二代PS,其强大的消光系数(ξmax>105 m-1cm-1)、长的最大吸收波长(λmax>670nm)以及可控的光物理和光化学性质使Pc成为能在PDT领域应用的合适候选。目前已经有四种酞菁偶联物进入临床试验阶段,它们分别是Photosens®、Pc4、CGP55847(ZnPc)和Photocyanine(福大赛因)。随着对结构的修饰,Pcs的各种功能被逐渐开发出来。然而,由于高度结合的疏水骨架,Pcs通常表现出较差的亲水性,并倾向于在水或生理溶液中聚集,导致三重态产率降低和活性氧生成减少。到目前为止,改善Pcs亲水性的方法主要有两种,一种是改变其分子结构,如设计离子型Pcs(包括阴离子、阳离子和两性离子)、聚乙二醇化Pcs,氨基酸或肽修饰的Pcs和碳水化合物取代的Pcs。另一个是构建纳米给药系统。在这个方向上,包括脂质体、纳米球和聚合物胶束在内的各种基质材料已与Pcs结合,以获得PS复合物,从而提高Pcs的溶解性和稳定性。尽管这些方法在一定程度上改善了水溶性,他们中的许多例子仍然无法避免Pc的聚集。他们的肿瘤选择性也不理想。正常组织(如肝脏)中的富集和肿瘤摄取不足不可避免的限制了其治疗效果。此外,大多数纳米给药系统存在制备繁琐、生物相容性差、潜在毒性等副作用,阻碍了其临床应用的发展。除此之外,大多数阴离子PSs(如磺酸阴离子PSs)因清除速度快和全身毒性低而受到青睐。然而,细胞摄取的不良不可避免地限制了其治疗效果。因此,开发一种安全有效的策略来同时提高Pcs的亲水性、减少聚集性、增加光敏活性以及细胞摄取能力并增强肿瘤靶向性仍然是一个巨大的挑战。
发明内容
本发明的目的在于提供一种能够克服在生理环境中聚集的、较高靶向性的以及能用于光动力治疗的光敏剂药物,具体提供一系列具有双摄取通路的轴向磺酸基修饰酞菁硅及其与白蛋白复合物的制备方法和在光动力治疗药物的应用,属于光敏剂光动力治疗药物领域。本发明所制得的具有双摄取通路的轴向磺酸基修饰酞菁硅光动力活性高、原料易得、制备简便,在生理体系中不易聚集,且能有效与白蛋白结合解聚。酞菁-白蛋白复合物具有稳定性高、细胞摄取率高、细胞光毒性高、体内靶向性好和体内代谢速度快等优点,可应用于光动力治疗。
为实现上述目的,本发明采用如下技术方案:
本发明的目的之一是保护一种具有双摄取通路的轴向磺酸基修饰酞菁硅,其能与白蛋白结合形成超分子复合物。
所述具有双摄取通路的轴向磺酸基修饰酞菁硅具体为四磺酸基酞菁硅,其结构式为:
本发明的目的之二是保护上述具有双摄取通路的轴向磺酸基修饰酞菁硅的制备方法,其中:
(1)以轴向二胺基取代酞菁硅和苯甲醛-2,4-二磺酸钠为反应物,使用分子筛净化过N,N-二甲基甲酰胺为溶剂,室温45 ℃下搅拌反应24 - 60小时,通过薄层色谱法监控反应终点,生成相应的具有双摄取通路的轴向磺酸基修饰酞菁硅(式1),进而通过溶剂法或者色谱法纯化目标产物;其中,所用轴向二胺基取代酞菁硅和苯甲醛-2,4-二磺酸钠的投料摩尔比为1:4~6;轴向二胺基取代酞菁硅的结构式为:,其制备方法为:在氮气保护下,将二氯硅酞菁、NaH和对羟基苯乙胺在无水甲苯中在110℃下混合搅拌48 h。在真空中蒸发溶剂后,将所得固体溶解在二氯甲烷中并离心,然后收集上清液,用去离子水冲洗上清液三次,溶剂在减压下蒸发,获得蓝色产物即轴向二胺基取代酞菁硅。
(2)以轴向二胺基取代酞菁硅和苯甲醛-2,4-二磺酸钠为反应物,使用分子筛净化过N,N-二甲基甲酰胺为溶剂,加入相应的三乙酰氧基硼氢化钠为还原剂,室温45 ℃下搅拌反应48 - 60小时,通过薄层色谱法监控反应终点,生成相应的具有双摄取通路的轴向磺酸基修饰酞菁硅(式2),进而通过溶剂法或者色谱法纯化目标产物。其中,所用轴向二胺基取代酞菁硅、三乙酰氧基硼氢化钠和苯甲醛-2,4-二磺酸钠的投料摩尔比为1:10:4~6。
本发明的目的之三是保护上述具有双摄取通路的轴向磺酸基修饰酞菁硅的白蛋白超分子复合物的制备方法及用于制备光动力药物。所述光动力药物能通过分子和超分子方法调控聚集,具有在生理环境中进一步解聚、产生高的活性氧的能力。另外,该光动力药物能通过磺酸阴离子与白蛋白介导的双摄取通道被肿瘤细胞摄取,并具有极高的光细胞毒性,同时能提高在体肿瘤靶向性。
所述光动力药物,或称光敏药物制剂,又称为光敏剂,可用于光动力治疗、光动力诊断或光动力消毒。所述光动力治疗可以是恶性肿瘤的光动力治疗,或是白血病的骨髓体外光动力净化治疗,或是非癌症疾病的光动力治疗,如真菌感染、细菌感染、口腔疾病、黄斑变性眼病、动脉硬化、创伤感染、皮肤病、病毒感染。所述光动力消毒可以是血液或血液衍生物的光动力灭菌净化,或是水的光动力灭菌消毒,或是医用或生活用器具的光动力消毒。
本发明所述具有双摄取通路的轴向磺酸基修饰酞菁硅-白蛋白复合物在光动力治疗、光动力诊断、光动力消毒和光动力降解污染物中的应用,需配套适宜的光源,所述适宜的光源可以由普通光源连接合适的滤光片来提供或由特定波长的激光或LED灯或其他灯源来提供,光源的波长范围为680~710 nm。
本发明的有益效果和突出优势是:
(1)酞菁类化合物作为新一代光敏剂可以应用于PDT中,通过将轴向胺基修饰酞菁硅与磺酸基的共价偶联制备出了酞菁硅与磺酸基的偶联物。细胞实验证明,本发明所述的四磺酸基酞菁硅可通过阴离子通路和白蛋白介导的双重途径被HepG2细胞吸收,从而增强细胞摄取,并最终导致极高的光细胞毒性,IC50值为2.0 nM。然而,目前文献报道的其他磺酸基酞菁不具备阴离子摄取通道,这是由于OATP的结构选择性导致的。这是首次发现阴离子酞菁通过OATP途径摄取,也是首个发现的光敏剂通过分子和超分子介导的双重摄取途径摄取。
(2)这种磺酸基修饰酞菁硅还可以与牛血清白蛋白(BSA)在水中形成的超分子复合物。超分子复合物的形成,可使磺酸基修饰酞菁硅基本以单体形式存在,有利于光动力活性的发挥。
(3)本发明所提供的四磺酸基酞菁硅与白蛋白形成的超分子复合物具有较高肿瘤靶向性和光动力抑制肿瘤生长的效应。
(4)本发明所得到的磺酸基修饰酞菁硅,制备过程操作简单、性质稳定、便于储存、有利于在工业生产中大批量的制备,产业化前景良好。
具体实施方式
为了使本发明所述的内容更加便于理解,下面结合具体实施方式对本发明所述的技术方案做进一步的说明,但是本发明不仅限于此。
实施例1
(1)轴向对称二胺基取代酞菁硅()的制备:
具体步骤如下:
在氮气保护下,将二氯硅酞菁(100.0 mg,0.2 mM)、NaH(32.0 mg)和对羟基苯乙胺(90.0 mg,0.7 mM)在无水甲苯(30 mL)中的混合物在110℃下搅拌48 h。在真空中蒸发溶剂后,将所得固体溶解在二氯甲烷中并离心,然后收集上清液。用去离子水冲洗上清液三次。溶剂在减压下蒸发。获得蓝色产物(80.0 mg),产率61.37 %。
表征数据:1H NMR (500Hz, Chloroform-d, δ): 9.61 - 9.62 (m, 8H, Pc -Hα), 8.35 - 8.36 (m, 8H, Pc -Hβ), 5.37 (d, J = 5.0 Hz, 4H; 2-H), 2.37(t, J =5.0 Hz, 3H; 1-H), 2.25 (t, J = 10.0 Hz, 4H; 4-H), 0.86 (t, J = 10.1 Hz, 4H;3-H).
HRMS (ESI) m/z: calcd for C48H36N10O2Si [M+H]+ 813.2865, found 813.2867.Relative error: 0.25 ppm.
(2)轴向四磺酸基修饰酞菁硅()的制备:将上述轴向对称二胺基取代酞菁硅(50.0 mg, 0.1 mM)和苯甲醛-2,4-二磺酸钠 (96.2mg,0.3 mM)在 DMSO 中的混合物在室温下搅拌 36 小时。真空蒸发溶剂后,产物通过硅胶柱色谱纯化,使用二氯甲烷和甲醇(4:1,v/v)作为洗脱剂,收集蓝绿色条带。然后在Bio-Beads S-X3柱和Bio-Beads S-X1柱上使用DMF作为洗脱剂的尺寸排阻色谱法纯化粗产物。收集蓝绿色条带,减压蒸馏溶剂得粗品。最后,将粗产物溶解在二氯甲烷(5 mL)中并倒入正己烷(100 mL)中,过滤收集产物并真空干燥,得到蓝色固体(14.9 mg),产率17.16 %。
表征数据:1H NMR (500 MHz, Methanol-d4, δ): 9.68 – 9.58 (m, 8H, Pc -Hα), 8.93 (s, 2H, 5-H), 8.45 (m, 8H, Pc -Hβ), 7.99 (s, 2H; 8-H), 7.85 (d, J =5.1 Hz, 2H; 7-H), 7.71 (d, J = 5.0 Hz, 2H; 6-H), 5.52 – 5.43 (d, J = 5.0 Hz,4H, 2-H), 3.08 – 2.93 (t, J = 7.7 Hz, 4H, 4-H), 2.39 – 2.28 (d, J = 5.0 Hz,4H, 1-H), 2.06 (t, J = 7.4 Hz, 4H; 3-H).
HRMS (ESI) m/z: calcd for C62H40N10Na4O14S4Si [M-3Na]3- 442.3656, found442.3771. Calcd for C62H40N10Na4O14S4Si [M-4Na]4- 326.0242, found 326.0335.Relative error: 2.6 ppm。
实施例2
目标四磺酸基酞菁硅偶联物()的制备:将实施例一所述轴向对称二胺基取代酞菁硅(50.0 mg,0.1 mM),三乙酰氧基硼氢化钠(20mg)和苯甲醛-2,4-二磺酸钠(96.2 mg,0.3 mM)在 DMSO 中的混合物在室温下搅拌 36 小时。真空蒸发溶剂后,产物通过硅胶柱色谱纯化,使用二氯甲烷和甲醇(4:1,v/v)作为洗脱剂,收集蓝绿色条带。然后在Bio-Beads S-X3柱和Bio-Beads S-X1柱上使用DMF作为洗脱剂的尺寸排阻色谱法纯化粗产物。收集蓝绿色条带,减压蒸馏溶剂得粗品。最后,将粗产物溶解在二氯甲烷(5 mL)中并倒入正己烷(100 mL)中,得到蓝色固体。过滤收集产物并真空干燥,得到蓝色固体(7.1 mg),产率8.16%。
表征数据:1H NMR (400MHz, DMSO-d6, δ): 9.58 – 9.73 (m, 8H, Pc -Hα),8.47 – 8.60 (m, 8H, Pc -Hβ), 8.05 (s, 2H, 9-H), 7.59 (d, J = 7.5 Hz, 2H; 8-H), 7.22 (d, J = 8.0 Hz, 2H; 7-H), 5.47 (d, J = 8.2 Hz, 4H; 2-H), 4.13 (s,4H, 6-H), 2.28 (d, J = 8.2 Hz, 4H; 1-H), 2.11 – 2.22 (t, J = 8.0 Hz, 4H, 4-H), 1.89 – 2.02 (t, J = 8.0 Hz, 4H, 3-H).
HRMS (ESI) m/z: calcd for C62H44N10O14S4Si [M-4Na+2H]2- 655.0918, found655.0928. Relative error: 1.53 ppm.
实施例3
(1)轴向对称二哌啶取代酞菁硅()的制备:在氮气保护下,将二氯硅酞菁(100mg, 0.2 mM),4-哌啶乙醇(0.212g,1.64 mM)和1ml吡啶溶于35ml无水甲苯中搅拌,110ºC反应24小时。真空旋转蒸发去除溶剂,将所得固体溶解在二氯甲烷中并离心,然后收集上清液。用去离子水冲洗上清液三次。溶剂在减压下蒸发。获得蓝色产物二(4-哌啶)乙氧基酞菁硅(62.0 mg),产率47.57%。
表征数据:1H NMR (400 MHz, DMSO-d6) δ 9.63 (m, 8H, Pc-Hα), 8.34 (m, 8H,Pc-Hβ), 1.27 (m, 4H), 0.88 (m, 2H), -0.59 (m, 2H), -0.49 (m, 8H), -1.64 (m,4H), -2.11 (m, 4H).
HRMS (ESI) m/z: calcd for C46H44N10O2Si [M+H]+ 797.3429, found 797.3451.Relative error: 2.76 ppm.
将上述案例二的轴向对称二哌啶取代酞菁硅(50mg, 0.063mmol)和溴代丙磺酸钠(0.142g,0.315mmol)、碳酸钾(0.088g,0.315mmol)溶于25mL DMF中搅拌,70°C反应24小时。真空旋转蒸发去除溶剂。粗产物通过硅胶柱纯化,使用DMF为洗脱剂,收集第一带。旋蒸至干,用少量水和DMSO溶解过G25凝胶,用水冲去第一带蓝色酞菁带,再用水(可用水和乙醇的混合溶液)收集留下来的蓝绿色酞菁带,旋蒸至干,干燥得蓝绿色粉末状产物15mg,产率48%。
表征数据:1H NMR (DMSO-d6, 400MHz, ppm): δ9.60~9.81 (m, 8H, Pc-Hα), δ8.43~8.63 (m, 8H, Pc-Hβ), δ2.17~2.32 (t, 4H, 8-H), δ1.81~2.16 (m, 8H, 5-H), δ1.38~1.51 (t, 4H, 6-H), δ1.18~1.29 (s, 2H, 3-H), δ0.72~1.01 (s, 4H, 7-H), δ-0.80~-0.42 (m, 8H, 4-H), δ-1.75~-1.58 (m, 4H, 2-H), δ-2.16~-2.08 (t, 4H, 1-H).
HRMS (ESI) m/z: calcd for C52H52N10Na2S8O2Si [M-2Na]2- 519.1669, found519.1674. Relative error: 0.96 ppm.
实施例4
(1)轴向对称二哌嗪取代酞菁硅()的制备:在氮气保护下,将二氯硅酞菁(100mg,0.2 mM),1-(2-羟乙基)哌嗪(0.212g,1.64mmol)和1ml吡啶溶于35ml无水甲苯中搅拌,110ºC反应24小时。真空旋转蒸发去除溶剂,将所得固体溶解在二氯甲烷中并离心,然后收集上清液。用去离子水冲洗上清液三次。溶剂在减压下蒸发。获得蓝色产物二[1-(2-羟乙基)哌嗪]酞菁硅(65.2 mg),产率49.90%。
表征数据:1H NMR (500 MHz, Methanol-d4) δ 9.70 m, (8H, Pc-Hα), 8.47 (m,8H, Pc-Hβ), 2.10 (t, J = 5.2 Hz, 8H), 0.90 – 0.85 (m, 2H), 0.61 (t, J = 5.1Hz, 8H), -0.48 (s, 4H), -1.89 (t, J = 5.3 Hz, 4H).
HRMS (ESI) m/z: calcd for C44H42N12O2Si [M+H]+ 798.3351, found 798.3374.Relative error: 2.88 ppm.
将轴向对称二哌嗪取代酞菁硅(50mg, 0.063mmol)和溴代丙磺酸钠(0.142g,0.315mmol)、碳酸钾(0.088g,0.315mmol)溶于25ml DMF中搅拌,70°C反应24小时。真空旋转蒸发去除溶剂,干燥得蓝色粗产物。用水和DMSO的混合溶液溶解,过G25水凝胶,收集第二带蓝绿色酞菁带,冻干干燥,得产物13.5mg,产率48%。
表征数据:1H NMR (400Hz, DMSO-d6, δ): 9.75 - 9.76 (m, 8H, Pc -Hα),8.55 - 8.56 (m, 8H, Pc -Hβ), 2.23 - 2.25 (t, J = 8.0 Hz, 4H, 7-H), 1.87 -1.92 (t, J = 20.0 Hz, 4H, 5-H), 1.15 - 1.55 (m, 12H, 4,6-H), 0.25 - 0.27 (t,J = 8.0 Hz, 8H, 3-H), -0.70 - -0.75 (t, J = 20.0 Hz, 4H, 2-H), -2.02 - -2.00(t, J = 8.0 Hz, 4H, 1-H).
HRMS (ESI) m/z: calcd for C50H52N12Na2O8S2Si [M-2Na+2H] 1042.3393, found1042.3385. Relative error: 0.77 ppm.
实施例5
周环取代磺酸取代酞菁锌(简称为ZnPcS1、ZnPcS2、ZnPcS4)的制备:
参考已发表文章 J. Am. Chem. Soc. 2019, 141, 1366-1372合成了ZnPcS1;参考已发表文章 Theranostics 2019, 9, 6412-6423合成了ZnPcS2和ZnPcS4。
实施例6
通过荧光共聚焦显微镜方法,比较实施例1-2的四磺酸基酞菁硅(SiPcS4-1和SiPcS4-2)、实施例3-5所述磺酸基修饰酞菁(SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4)、以及常见水溶性光敏剂Ce6和MB的细胞摄取:
将本发明实施例1-5所述制备的磺酸酞菁偶联物溶于DMF中,配置成1 mM的母液,然后在细胞培养基进行稀释,制成一定浓度的光敏药剂。在DMEM细胞培养基(0.4 mL)中,将约5 x 104 HepG2细胞悬浮液接种在共聚焦培养皿上,并在37 ℃下用5% CO2培养过夜。去除培养基后,加入本发明实施例1-5所述制备的磺酸酞菁偶联物:SiPcS4-1(100 nM)、SiPcS4-2(100 nM)、SiPcS2-1(100 nM)、SiPcS2-2(100 nM)、ZnPcS1(4 μM)、ZnPcS2(4 μM)、ZnPcS4(4 μM)、MB(4 μM)和Ce6(4 μM)。培养2h并去除培养基后。用PBS冲洗细胞,使用莱卡TCS SPE共聚焦显微镜成像。光敏剂在635nm处激发,在640-750nm处监测。记录细胞内平均荧光强度(每个样本共30个细胞),计算各实验组的相对荧光强度。结果如下表所示,表明SiPcS4-1显示出最强的细胞内荧光强度,表明该化合物具有最强的细胞摄取能力,显著强于结构相似的磺酸基修饰酞菁和常见的水溶性光敏剂MB与Ce6。
实施例7
细胞摄取通路研究
通过荧光共聚焦显微镜方法,研究实施例1-2的四磺酸基酞菁硅(SiPcS4-1和SiPcS4-2)的细胞摄取通路:
将SiPcS4-1和SiPcS4-2溶于DMF中,配置成1 mM的母液,然后在细胞培养基进行稀释,制成一定浓度的光敏药剂。在DMEM细胞培养基(0.4 mL)中,将约5 x 104 HepG2细胞悬浮液接种在共聚焦培养皿上,并在37 ℃下用5% CO2培养过夜。去除培养基后,分别用各种摄取抑制剂预处理细胞,包括阿米洛利(肌动蛋白抑制剂,20 μg/mL,30分钟)、氯丙嗪(网格蛋白抑制剂,10 μg/mL,30分钟)、甲基-β-环糊精(MβCD,小窝抑制剂,7.5 mM,30分钟)和磺溴酞钠(BSP,竞争性OATP转运蛋白抑制剂,250 mM,5分钟)。然后,分别加入SiPcS4-1和SiPcS4-2(100 nM)共培养30分钟。用PBS冲洗细胞,并使用莱卡TCS SPE共聚焦显微镜成像。酞菁在635nm处激发,在640-750nm处监测。记录细胞内平均荧光强度(每个样本共30个细胞),计算各实验组的相对荧光强度。实验结果如下表所示,SiPcS4-1和SiPcS4-2的细胞摄取不受到肌动蛋白抑制剂和网格蛋白抑制剂的抑制,而受到小窝抑制剂和竞争性OATP转运蛋白抑制剂的抑制。说明SiPcS4-1和SiPcS4-2是通过细胞内吞小窝途径(白蛋白摄取途径)以及细胞OATP阴离子转运途径被细胞摄取的。
实施例8
通过荧光共聚焦显微镜方法,研究实施例1-5的磺酸基酞菁(SiPcS4-1、SiPcS4-2、SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4)的细胞小窝途径以及细胞OATP阴离子转运途径的摄取通路:
将本发明实施例1-5所述制备的磺酸基酞菁偶联物溶于DMF中,配置成1 mM的母液,然后在细胞培养基进行稀释,制成一定浓度的光敏药剂。在DMEM细胞培养基(0.4 mL)中,将约5 x 104 HepG2细胞悬浮液接种在共聚焦培养皿上,并在37 ℃下用5% CO2培养过夜。去除培养基后,分别用甲基-β-环糊精(MβCD,小窝抑制剂,7.5 mM,30分钟)和磺溴酞钠(BSP,竞争性OATP转运蛋白抑制剂,250 mM,5分钟)摄取抑制剂预处理细胞。然后,分别加入100 nM的SiPcS4-1、SiPcS4-2和4 μM 的SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4共培养30分钟。用PBS冲洗细胞,并使用莱卡TCS SPE共聚焦显微镜成像。酞菁在635nm处激发,在640-750nm处监测。记录细胞内平均荧光强度(每个样本共30个细胞),计算各实验组的相对荧光强度。实验结果如下表所示。
细胞摄取机制实验证明,经甲基-β-环糊精(MβCD,小窝途径抑制剂)或磺基溴酞(BSP,有机阴离子转运多肽(OATP)抑制剂)预处理的SiPcS4-1和SiPcS4-2给药的HepG2细胞的荧光被抑制,表明SiPcS4-1和SiPcS4-2的细胞摄取是由小窝途径和OATP途径介导的。然而,SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4给药的HepG2细胞却没有显示出通过OATP通道摄取的趋势。正是由于缺少了这种有效的摄取通道,导致SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4的细胞摄取显著低于SiPcS4-1和SiPcS4-2。由于细胞小窝途径是白蛋白从血液有效摄取和转运至间质所必需的。而OATP转运体在多种癌细胞中过度表达,并且能够在膜上转运许多阴离子基质。因此,SiPcS4-1和SiPcS4-2可通过磺酸阴离子和白蛋白介导的双细胞途径被HepG2细胞吸收,从而显著提高其细胞摄取效率。
实施例9
用分子模拟计算验证实施例1-2所述四磺酸酞菁硅(SiPcS4-1和SiPcS4-2)与OATP转运蛋白的可能结合构象与潜在结合机制:
为了探索OATP途径的结构选择性,进行了分子对接计算以阐明人类OATP转运蛋白(PDB ID:6S4M)对SiPcS4-1和SiPcS4-2的结构选择性的潜在机制。在所有结合模型中,四磺酸酞菁硅都良好的插入OATP转运蛋白的中央口袋。具体而言,SiPcS4-1和SiPcS4-2的带负电的磺酸盐部分和带正电的残基(R153)之间建立了离子键,表明SiPcS4-1和SiPcS4-2的磺酸盐基团和OATP转运蛋白具有强烈的亲和力。此外,SiPcS4-1和SiPcS4-2可以与Q264和S374建立氢键,以与R153形成特定的结合囊,这可能有助于其与OATP转运蛋白更稳定和更好的结合。相反,实施例3-5所述磺酸基酞菁(SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4)由于静电相互作用减弱或结合位置不当而无法装入袋中。因此,与OATP转运蛋白特异性结合口袋的相互作用被认为是激活OATP通路的结构决定因素。
实施例10
测试实施1-2所述磺酸基修饰酞菁硅(SiPcS4-1和SiPcS4-2)在不同溶剂中的分散状态及其与BSA的结合实验:
将SiPcS4-1和SiPcS4-2溶于DMF中,配置成1 mM的母液,利用紫外-可见吸收光谱仪测试它们在不同溶液中的分散状态,不同的溶液包括N, N-二甲基甲酰胺、PBS、和10% FBS。然后向BSA的水溶液中加入SiPcS4-1和SiPcS4-2,测试其与BSA的结合常数。
测试的结果表明,SiPcS4-1和SiPcS4-2在N, N-二甲基甲酰胺中的Q带为强而尖的峰,说明在N, N-二甲基甲酰胺中所得磺酸酞菁硅偶联物是以单体的形式存在。但是在纯水中呈现出宽而矮的Q带,说明在纯水中所得磺酸酞菁硅偶联物多数以聚集体形式存在。然而,在10% FBS中SiPcS4-1和SiPcS4-2呈现出部分解聚的单体形式,说明能在FBS中部分解聚。因此,继续测定了FBS中的BSA与SiPcS4-1和SiPcS4-2的结合作用。参照J. Am. Chem.Soc. 2019, 141, 1366−1372的测定方法,测得SiPcS4-1和SiPcS4-2与BSA结合常数分别是494311和601174,酞菁与BSA的结合比分别是1.012和1.324。
实施例11
四磺酸基酞菁硅偶联物(SiPcS4-1)-白蛋白超分子复合物的制备:
利用本发明SiPcS4-1制备SiPcS4-1-BSA超分子复合物的光动力治疗的药物的方法是:先将本发明所述制备的SiPcS4-1与DMF溶解配置成1 mM的母液,再以与BSA浓度比1:1的比例加入等浓度的含有BSA的PBS中。配置成一定浓度的SiPcS4-1-BSA超分子复合物。
实施例12
离体光动力抗癌活性研究:
实施例1-5所述的磺酸基酞菁(SiPcS4-1、SiPcS4-2、SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2和ZnPcS4)、实施例11所述四磺酸基酞菁硅-白蛋白超分子复合物(SiPcS4-1-BSA)和已知光敏剂MB和Ce6各配置成1 mM的母液,之后再进行稀释,制成一定浓度的光敏药剂。测试它们在人肝癌细胞HepG2中的光毒性。另外,对比测试实施例1的四磺酸基酞菁硅(SiPcS4-1)和实施例11所述四磺酸基酞菁硅-白蛋白超分子复合物(SiPcS4-1-BSA)的暗毒性。
细胞光毒性与暗毒性测定:将HepG2细胞(约1×104个细胞/孔)在37 ℃的加湿5%CO2气氛中在96孔板中维持过夜。然后抽吸并丢弃旧培养基,并将100 μL/孔的SiPcS4-1、SiPcS4-2、SiPcS2-1、SiPcS2-2、ZnPcS1、ZnPcS2、ZnPcS4、SiPcS4-1-BSA、MB和Ce6加入所需的浓度梯度。培养2h并去除培养基后,用PBS冲洗细胞,并用100 μL培养基重新孵育。然后在室温下将光照0.5小时。光源包括一个500 W卤素灯、一个用于冷却的水箱和一个610 nm的彩色玻璃过滤器。光功率为15 mW/cm2,总光量为27 J/cm2。MTT法测定细胞活力。光照后,细胞在37 ℃和5% CO2下培养过夜。PBS中的MTT溶液向每个孔中添加2.5 mg/mL,然后在相同条件下培养4 h。取出细胞后,将二甲基亚砜添加到每个孔中测定吸光度。在测定吸光度(490nm)之前,在环境温度下,在微孔板读取器(Tecan M200Pro)的96孔板上搅拌20 s。然后通过以下方程式检测细胞活力:
式中,Ai是第i个数据的吸光度(i=1,2,…,n),Ācontrol是不含Pc的对照孔的平均吸光度,n (3)是数据点的数目。
通过考察所得磺酸基酞菁、四磺酸基酞菁硅-白蛋白超分子复合物或MB和Ce6的浓度和细胞存活率的量效关系,获得在光照条件下的半致死浓度(EC50,即光照下杀死50%癌细胞所需的药物浓度)总结于下表。
实验结果表明:与其他磺酸基酞菁和已知光敏剂MB和Ce6相比,SiPcS4-1和SiPcS4-2在光照条件下的EC50显著降低,说明了SiPcS4-1和SiPcS4-2具有潜在的优异的离体治疗效果。另外,获得SiPcS4-1-BSA在无光照条件下的半致死浓度(TC50,即黑暗条件下杀死50 %癌细胞所需的药物浓度)为1821 nM,相较于SiPcS4-1在无光照条件下的TC50 (1540 nM)有所增加,说明了实施例11所述的方法制备的SiPcS4-1-BSA具备更弱的暗毒性,具有更安全的治疗窗口与更好的生物相容性。
实施案例13
将实施例11所述四磺酸基酞菁硅-白蛋白超分子复合物(SiPcS4-1-BSA)配置成1mM的PBS溶液,再用PBS进行稀释,制成浓度为200 μM的光敏药剂溶液。测试其对种有肝癌细胞(H22)的固体瘤的KM小鼠的荧光成像情况。对种有H22的荷瘤小鼠尾静脉注射100 μL浓度为200 μM的SiPcS4-1-BSA。使用小动物荧光成像仪监测SiPcS4-1-BSA在小鼠肿瘤部位的富集情况,24小时后对小鼠进行解剖,使用小动物荧光成像仪监测药物在小鼠各个组织器官的分布情况。详细实验步骤参见ACS Appl. Mater. Interfaces 2019, 11, 36435−36443。
在体荧光实验结果表明:上述SiPcS4-1-BSA显示出了较高的肿瘤靶向性。在静脉注射后刚开始全身各部几乎没有任何的荧光显示,一小时之后在体内迅速循坏,随后逐渐在肿瘤部位富集,并且能在肿瘤部位长时间保留,显示出较高的靶向效果。24小时后对注射SiPcS4-1-BSA的小鼠进行解剖包括小鼠肿瘤以及肿瘤附近的皮肤,发现除了小鼠的肿瘤部位可以观察到较为明显的荧光,而在其体内的其它各个器官组织中未发现明显的多余肿瘤部位的药物残留情况。这些实验结果说明本发明所述的SiPcS4-1-BSA在小鼠体内具有较好在肿瘤部位富集,而且由于内源性的制备方式导致药物在正常组织中更容易代谢而不残留,具有较好的生物安全性。
实施案例14
测试实施例11所述四磺酸基酞菁硅-白蛋白超分子复合物(SiPcS4-1-BSA)对种有肝癌细胞(H22)的固体瘤的KM小鼠的肿瘤抑制率:
取已种植皮下肿瘤的KM小鼠,分为4组给药(SiPcS4-1-BSA给药+激光组,SiPcS4-1-BSA给药组,盐水+激光组,盐水组),每组5只;待肿瘤长到60 - 100 mm3大小时,静脉注射100 μL浓度为200 μM的SiPcS4-1-BSA的PBS溶液。分别于24小时后,将小鼠麻醉,用激光685± 5 nm激光照射(光照强度31 mW/cm2,光照时间5分钟)。继续饲养小白鼠,隔日观察小鼠情况。并于第7日再次进行静脉给药,第8日再次进行激光照射(光照强度31 mW/cm2,光照时间5分钟)。期间隔日测量小白鼠的体重,并用游标卡尺测量小白鼠的长直径与短直径,一共测量14天。
结果显示,经过14天的实验,SiPcS4-1-BSA给药组,盐水+激光组和盐水组的小鼠肿瘤均增长了大约12倍,在使用685 nm(光照强度31 mW/cm2)的激光对SiPcS4-1-BSA给药的小鼠的肿瘤部位进行连续照射5 min后,小鼠肿瘤显示了有效的抑制肿瘤生长的作用,显示出90%的抑瘤率(p<0.001)。这说明按照本发明所述的SiPcS4-1-BSA在光动力治疗下表现出较为优异的抗肿瘤活性。另外,治疗组小鼠在14天内体重呈增加趋势,说明SiPcS4-1-BSA对小鼠没有明显毒性,具有良好的生物相容性。以上实验表明,本发明所提供的SiPcS4-1-BSA是一种具有高的光动力活性和生物安全性的靶向抗肿瘤药物。
以上所述仅为本发明的较佳实施例,凡依本发明申请专利范围所做的均等变化与修饰,皆应属本发明的涵盖范围。
Claims (7)
3.根据权利要求2所述的制备方法,其特征在于:所用轴向二胺基取代酞菁硅和苯甲醛-2,4-二磺酸钠的摩尔比为1:4~6。
5.根据权利要求4所述的制备方法,其特征在于:所用轴向二胺基取代酞菁硅、三乙酰氧基硼氢化钠和苯甲醛-2,4-二磺酸钠的摩尔比为1:10:4~6。
6.一种如权利要求1所述的具有双摄取通路的轴向磺酸基修饰的酞菁硅在制备光动力药物中的应用。
7.根据权利要求6所述的应用,其特征在于:将具有双摄取通路的轴向磺酸基修饰酞菁硅与牛血清白蛋白复合得到酞菁-白蛋白复合物,用于制备光动力药物。
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