Tubulin-Binding Upconversion Nanoparticles for Breast-Cancer Imaging and Therapy

描述（由申请人提供）：正常细胞分裂需要形成有丝分裂纺锤体，其与微管（MT）组装直接相关。因此，如果微管蛋白结合剂抑制MT组装，则有丝分裂纺锤体功能将受到干扰，导致在有丝分裂的中期/后期转换时抑制细胞分裂。因此，微管蛋白结合剂是一种抗癌药物，可以通过干扰MT组装来抑制细胞分裂并导致癌细胞死亡。然而，目前结合微管蛋白的抗癌药物无法特异性识别癌细胞并作为成像探针。本实验室最近利用噬菌体展示技术成功地鉴定了两种类型的肽：微管蛋白结合肽和SKBR-3乳腺癌细胞靶向/内化肽。此外，我们还开发了镧系离子掺杂的上转换纳米颗粒（UCNPs）的合成及其作为癌细胞成像探针的应用。与诸如广泛使用的量子点（QD）的降频转换纳米颗粒相比，UCNP可以被诸如近红外（NIR）光（例如，980 nm）以发射较短波长的光（例如，绿色光）。由于它们能够被NIR光激发，NIR光可以相对深入地穿透细胞和组织，UCNP可以用于细胞/组织成像，而不会引起生物分子、细胞和组织的自发荧光或光损伤。该项目基于这些成功，将微管蛋白结合肽和癌症靶向/内化肽结合到UCNP表面上。我们的假设是，核-壳上转换纳米粒子（稀土掺杂？NaYF 4：Yb，Er上转换（作为核，二氧化硅作为壳）与微管蛋白结合肽和SKBR-3乳腺癌细胞靶向/内化肽两者缀合到表面将（1）识别乳腺癌细胞并被内化;（2）结合微管蛋白以中断细胞内MT组装，抑制细胞增殖并在体外和体内引起细胞死亡;和（3）使得能够在NIR激发下对癌细胞和肿瘤组织进行选择性荧光成像。我们将进行两个具体的目的：（1）目的1：评估在UCNP存在下的体外MT组装，其中微管蛋白结合肽和SKBR-3癌细胞靶向/内化肽均缀合至表面，以理解细胞靶向微管蛋白结合UCNP如何在体外中断MT组装。(2)目标二：评价体外SKBR-3乳腺癌细胞增殖和细胞周期以及SKBR-3细胞与细胞靶向微管蛋白结合UCNP体外或体内相互作用后的体外和体内靶向癌细胞成像和杀伤。该项目将通过开发靶向癌症成像和治疗药物来推进靶向癌症治疗和诊断。该项目开发的癌症靶向微管蛋白结合UCNP是多功能治疗诊断剂，可以靶向和杀死癌细胞，同时在癌细胞和肿瘤组织内进行荧光检测和跟踪。

DESCRIPTION (provided by applicant): Normal cell division needs the formation of a mitotic spindle apparatus, which is directly related to microtubule (MT) assembly. Thus, if the MT assembly is inhibited by tubulin-binding agents, the mitotic spindle functions will be perturbed, resulting in the inhibition of cell division at the metaphase/anaphase transition of mitosis. Therefore, tubulin-binding agents are anti-cancer drugs that can inhibit cell division by perturbing the MT assembly and cause cancer cell death. However, the current tubulin-binding anti-cancer drugs cannot specifically recognize cancer cells and serve as imaging probes. Our lab has recently used phage display technique to successfully identify two types of peptides: tubulin-binding peptides and SKBR-3 breast cancer cell-targeting/internalizing peptides. Moreover, we have developed expertise in the synthesis of lanthanide ion doped upconversion nanoparticles (UCNPs) and their utilization as cancer cell imaging probes. In contrast to the down-conversion nanoparticles such as the widely used quantum dots (QDs), the UCNPs can be excited by a longer wavelength light such as near infrared (NIR) light (e.g., 980 nm) to emit a shorter wavelength light (e.g., green light). Due to their ability to be excited by NIR light, whic can penetrate cells and tissues relatively deeply, UCNPs can be used for cell/tissue imaging without causing either autofluorescence from or photodamage to the biomolecules, cells and tissues. This project is based on these successes and will conjugate both tubulin-binding and cancer-targeting/internalizing peptides onto the surface of UCNPs. Our hypothesis is that core-shell upconversion nanoparticles (Rare-earth doped ?-NaYF4:Yb,Er upconversion nanocrystal as a core and silica as a shell) with both tubulin-binding and SKBR-3 breast cancer cell- targeting/internalizing peptides conjugated to the surface will (1) recognize the breast cancer cells and be internalized; (2) bind tubulins to interrupt intracellular MT assembly, inhibit cell proliferation and cause cell death in vitro and in vivo; and (3) enable the selective fluorescent imaging of the cancer cells and tumor tissues under NIR excitation. We will carry out two specific aims: (1) Aim 1: Evaluate the in vitro MT assembly in the presence of UCNPs with both tubulin-binding peptides and SKBR-3 cancer cell-targeting/internalizing peptides conjugated to the surface to understand how cell-targeting tubulin-binding UCNPs interrupt MT assembly in vitro. (2) Aim 2: Evaluate in vitro SKBR-3 breast cancer cell proliferation and cell cycle as well as in vitro and in vivo targeted cancer cell imaging and killing after SKBR-3 cells interact with the cell-targeting tubulin-binding UCNPs in vitro or in vivo. This project will advance the targete cancer treatment and diagnosis by developing targeted cancer imaging and therapeutic agents. The cancer-targeting tubulin-binding UCNPs developed in this project are multi-functional theranostic agents that can target and kill cancer cells and at the same time be fluorescently detected and tracked inside the cancer cells and tumor tissues.