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Targeting single-walled carbon nanotubes for the treatment of breast cancer using photothermal therapy.

机译：使用光热疗法靶向单壁碳纳米管以治疗乳腺癌。

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摘要

To develop a therapeutic system with cancer cell selectivity, the present study evaluated a possible specific and localized tumor treatment. Phosphatidylserine (PS) exposure on the external face of the cell membrane is almost completely exclusive to cancer cells and endothelial cells in the tumor vasculature. The human protein annexin V is known to have strong calcium-dependent binding to anionic phospholipids such as PS. This protein was studied for targeting single-walled carbon nanotubes (SWNTs) to the vasculature of breast tumors.;The synthesis of the protein annexin V, by a pET vector in Escherichia coli, constitutes the first phase of this study. Recombinant annexin V was purified from the cell lysate supernatant by immobilized metal affinity chromatography. The overall production of purified annexin V protein was 50 mg/L.;The binding ability of the protein annexin V was evaluated by determining the dissociation constant when incubated with proliferating human endothelial cells in vitro. The dissociation constant, Kd, was measured to be 0.8 nM, indicating relatively strong binding. This value of Kd is within the range reported in the literature.;Single-walled carbon nanotubes (SWNTs) were functionalized with annexin V using two intermediate linkers (containing FMOC and DSPE) resulting in stable suspensions. The SWNT and protein concentrations were 202 mg/L and 515 mg/L, respectively, using the linker with DSPE (average of nine preparations). The conjugation method that used the DSPE-PEG-maleimide linker allowed to successfully conjugate the SWNTs with final concentrations approximately five times higher than the linker containing FMOC. The conjugation method used has a non-covalent nature, and therefore the optical properties of the nanotubes were preserved.;The conjugate was also visually observed using atomic force microscopy (AFM), allowing to verify the presence of the protein annexin V on the surface of the nanotubes, with an height ranging between 2.5 to 5.0 nm. Confocal microscopy was used to prove the binding of the conjugates to human endothelial cells grown in vitro.;Proliferating endothelial cells were used to determine the optimal near-infrared (NIR) laser irradiation settings (energy density = 200 J/cm2), which would not induce cell cytotoxicity from the laser itself. A 2 hour incubation with the conjugate followed by a washing step and NIR irradiation (wavelength = 980 nm, power = 1 W/cm2, time = 200 seconds) was enough to induce significant cell death (≈55 %), without significant damage to the control samples.;Administration of the same conjugates i.v. in Balb/cJ female mice resulted in detectable accumulation of the SWNTs in the tumor tissues, with minimal retention in the kidneys 24 hours post-administration. A dosage of 0.82 mg/kg of SWNTs administered i.v. and followed one day later by a NIR irradiation (wavelength = 980 nm, power = 1 W/cm2, time = 175 seconds) led to complete disappearance of implanted 4T1 mouse mammary tumors for the majority of the animals from the treatment groups, within a few days. The combination of the photothermal therapy with a low dosage (50 mg/kg) of the immunoadjuvant cyclophosphamide, given 2 days before NIR irradiation, was also evaluated; this resulted in increased survival. Histological findings revealed the complete obliteration of the tumors treated from the original site, with complete regeneration of the skin epithelial layer and absence of cancer cells.;In conclusion, this research was successful in demonstrating that SWNTs could be targeted to the tumor vasculature in vivo and then could be heated by NIR irradiation to completely kill mouse mammary tumors implanted in immune-competent mice. There is evidence that the co-administration of the immunostimulant cyclophosphamide caused increased survival of the mice. It is recommended that future work be directed to exploring methods to increase the concentration of the SWNT-annexin V conjugate in the tumor and to evaluate the co-administration of other immunostimulants, either alone or in combination.
机译：为了开发具有癌细胞选择性的治疗系统，本研究评估了可能的特异性和局部肿瘤治疗。细胞膜外表面的磷脂酰丝氨酸（PS）暴露几乎完全排除了肿瘤脉管系统中的癌细胞和内皮细胞。已知人蛋白膜联蛋白V与阴离子磷脂（如PS）具有很强的钙依赖性结合。该蛋白质被研究用于将单壁碳纳米管（SWNTs）靶向乳腺肿瘤的脉管系统。大肠杆菌中pET载体合成膜联蛋白V构成了该研究的第一阶段。通过固定的金属亲和层析从细胞裂解物上清液中纯化重组膜联蛋白V。纯化的膜联蛋白V蛋白的总产量为50 mg / L。通过与增殖的人内皮细胞体外孵育确定离解常数，评估了膜联蛋白V的结合能力。测得解离常数Kd为0.8 nM，表明结合力较强。 Kd的这一值在文献报道的范围内。使用两个中间连接物（包含FMOC和DSPE），用膜联蛋白V对单壁碳纳米管（SWNT）进行功能化，从而得到稳定的悬浮液。使用带有DSPE的接头（九种制剂的平均值），SWNT和蛋白质浓度分别为202 mg / L和515 mg / L。使用DSPE-PEG-马来酰亚胺接头的偶联方法可以成功偶联SWNT，其最终浓度比含FMOC的接头高约五倍。所使用的偶联方法具有非共价性质，因此保留了纳米管的光学性质。;还使用原子力显微镜（AFM）肉眼观察了偶联物，从而可以验证表面上是否存在膜联蛋白V纳米管的高度在2.5至5.0nm之间。共聚焦显微镜用于证明结合物与体外培养的人内皮细胞的结合;增殖的内皮细胞用于确定最佳近红外（NIR）激光照射设置（能量密度= 200 J / cm2）不会从激光本身诱导细胞毒性。与结合物温育2小时，然后进行洗涤步骤和NIR辐照（波长= 980 nm，功率= 1 W / cm2，时间= 200秒）足以诱导显着的细胞死亡（约55％），而没有明显的损伤对照样品;相同的结合物的管理iv Balb / cJ雌性小鼠中的“小白鼠”导致SWNTs在肿瘤组织中可检测到的积累，给药后24小时在肾脏中的保留最小。静脉内施用0.82 mg / kg的SWNTs。然后一天后进行NIR照射（波长= 980 nm，功率= 1 W / cm2，时间= 175秒），导致治疗组中大多数动物的植入的4T1小鼠乳腺肿瘤完全消失。几天。还评估了在NIR照射前2天给予的光热疗法与低剂量（50 mg / kg）免疫佐剂环磷酰胺的组合；这导致生存期增加。组织学发现揭示了从原始部位完全消灭的肿瘤，皮肤上皮层的完全再生以及癌细胞的缺失。；总而言之，这项研究成功地证明了SWNTs可以在体内靶向肿瘤血管然后可以通过NIR辐射加热以完全杀死植入有免疫能力的小鼠的乳腺肿瘤。有证据表明，免疫刺激剂环磷酰胺的共同给药可增加小鼠的存活率。建议将来的工作应针对探索增加肿瘤中SWNT-annexin V偶联物浓度并评估其他免疫刺激剂单独或组合共同给药的方法。

著录项

  • 作者

    Neves, Luis Filipe Ferreira.;

  • 作者单位

    The University of Oklahoma.;

  • 授予单位 The University of Oklahoma.;
  • 学科 Engineering Biomedical.;Health Sciences Medicine and Surgery.;Nanotechnology.;Health Sciences Oncology.
  • 学位 Ph.D.
  • 年度 2012
  • 页码 202 p.
  • 总页数 202
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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