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

描述（由申请人提供）：正常的细胞分裂需要有丝分裂纺锤体的形成，这与微管（MT）组装直接相关。因此，如果MT组装受到微管蛋白结合剂的抑制，有丝分裂纺锤体功能将受到干扰，导致有丝分裂中期/后期转变时细胞分裂受到抑制。因此，微管蛋白结合剂是一种通过扰乱MT组装来抑制细胞分裂并导致癌细胞死亡的抗癌药物。然而，目前的微管蛋白结合抗癌药物不能特异性识别癌细胞，不能作为成像探针。我们的实验室最近使用噬菌体展示技术成功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上转换纳米颗粒（UCNPs）的合成技术，并将其用作癌细胞成像探针。与下转换纳米粒子（如广泛使用的量子点（QDs））相比，UCNPs可以被波长较长的光(如近红外（NIR）光（如980 nm）激发，发出波长较短的光（如绿光）。由于它们能够被近红外光激发，而近红外光可以相对深入地穿透细胞和组织，因此UCNPs可以用于细胞/组织成像，而不会引起生物分子、细胞和组织的自身荧光或光损伤。该项目以这些成功为基础，将微管蛋白结合和癌症靶向/内化肽结合到UCNPs表面。我们的假设是核壳上转化纳米粒子(稀土掺杂？- nayf4:Yb，Er上转换纳米晶体为核心，二氧化硅为外壳)结合微管蛋白结合和SKBR-3乳腺癌细胞靶向/内化肽偶联到表面将(1)识别乳腺癌细胞并内化；(2)结合微管蛋白阻断细胞内MT组装，抑制细胞增殖，导致细胞死亡；(3)在近红外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标：(1)目标1：评估在UCNPs存在下，微管蛋白结合肽和结合到表面的SKBR-3癌细胞靶向/内化肽的体外MT组装，以了解细胞靶向微管蛋白结合的UCNPs如何在体外中断MT组装。(2)目的2：体外评价SKBR-3乳腺癌细胞增殖和细胞周期，以及SKBR-3细胞在体外和体内与靶向细胞的微管蛋白结合UCNPs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肿瘤成像和治疗剂来推进肿瘤的靶向治疗和诊断。本项目开发的靶向肿瘤微管蛋白结合UCNPs是一种既能靶向并杀死癌细胞，又能在癌细胞和肿瘤组织内部进行荧光检测和跟踪的多功能治疗剂。

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.