Formulation of a targeted nanoparticle system for the treatment of chemoresistant breast cancer

摘要 癌细胞的多药耐药(MDR)的发展令人严重关注，限制了 抗癌药物的疗效，因此，乳腺癌治疗的失败。临床研究和 应用表明，尽管阿霉素具有潜在的抗癌作用，但它的毒性很高，其 长期应用可引起剂量依赖性不可逆性心肌病，严重心脏 毒性或肝脏损伤，从而限制了其在乳腺癌治疗中的应用。即使这种药物 是超高效的，如果它仍然造成非靶标毒性并损害非癌症细胞和组织， 这种药物不是治疗这种特殊疾病的良药。因此，更大的潜力 是否使用阿霉素作为抗癌治疗取决于靶向递送的可用性 载体，它不仅可以增强对癌细胞的杀灭作用，还可以最大限度地减少脱靶 对非癌细胞的毒性。这项研究的目的是提高阿霉素的递送 通过配制适配子标记的脂质体纳米粒递送系统，将携带和 将阿霉素特异性地输送到化疗耐药的HER-2+乳腺癌细胞。我们有 最近报道P-gp下调MDR1 P-gp(ABCB1基因)的核表达 特异性siRNA可增加阿霉素耐药乳腺癌细胞对阿霉素的转运。 然而，由于Dox是以自由药物溶液的形式提供的，没有将其封装成颗粒 对于靶向递送，它仍然对其他非癌细胞细胞产生毒性。有针对性地交付 抑制mdr1 P-gp、MRP或BCRP等多药耐药基因的siRNA可能会有所帮助 然而，为了使用我们在实验室开发的APT标记配方来规避MDR， 仍然有一些问题需要解决：(1)我们如何在 更具靶向性的方式使耐药的乳腺癌细胞药物增强 对癌细胞的毒性增加，而对非癌细胞的毒性最小？我们假设 一个有针对性的递送系统是最高要求，无论它是递送siRNA来沉默 化疗耐药基因或真正的化疗地毯，它将杀死癌细胞，而不会杀死非 癌细胞。为了解决化疗耐药性和靶外毒性，一种定向递送 需要开发具有创新性、可比性和可比性的阿霉素系统 将对其他非癌症细胞的毒性降至最低。以及(2)需要制定一项战略，以 确定靶向纳米粒是否会同时携带阿霉素和siRNA 或在不同的颗粒中获得最佳效果，防止化疗耐药和限制关闭- 靶标毒性。我们的假设是，分别提供阿霉素和MDR沉默的siRNA 与靶向纳米粒系统相比，可增强细胞毒性和抗肿瘤作用 一种同时输送药物和siRNA的靶向纳米颗粒系统。这一假设 将通过两个具体目标进行测试： 目的1：靶向阿霉素脂质体治疗HER-2阳性乳腺癌。 目标2：评估靶向纳米粒是否会同时携带阿霉素和siRNA 相同的颗粒或在不同的颗粒中获得最佳效果，防止化疗耐药和限制 偏离目标的毒性。

ABSTRACT The development of multidrug resistance (MDR) in cancer cells is of grave concern, limiting the efficacy of anticancer agents and, hence, the failure of breast cancer therapy. Clinical research and application revealed that in spite of its potential anticancer effects, doxorubicin is highly toxic, and its long-term application may cause dose-dependent irreversible cardiomyopathy, severe cardiac toxicity, or liver damage, thereby limiting its application in breast cancer treatment. Even if the drug is super-efficient, if it still causes off-target toxicity and damages non-cancerous cells and tissues, the drug wouldn’t be a great remedy to treat that particular disease. As such, the greater potential of using doxorubicin as anticancer therapeutic depends on the availability of a targeted delivery vehicle, which will not only enhance the killing of cancer cells but also minimize the off-target toxicity to non-cancerous cells. The goal of this study is to enhance the delivery of doxorubicin by formulating an aptamer-labeled liposomal nanoparticle delivery system that will carry and deliver doxorubicin specifically into chemoresistant Her-2+ breast cancer cells. We have recently reported that down regulating nuclear expression of MDR1 P-gp (ABCB1 gene) by P-gp specific siRNA could increase the delivery of doxorubicin to doxorubicin resistant breast cancer cells. However, since the Dox was delivered as a free drug solution without encapsulating it into a particle for targeted delivery, it still caused toxicity to other non-cancerous cells. The targeted delivery of siRNA to knockdown multi-drug resistant genes such as MDR1 P-gp, MRP or BCRP might be helpful to circumvent MDR using the apt-labeled formulations that we have developed in our lab, however, there are some questions that still need to be addressed (1) how can we deliver doxorubicin in a more targeted fashion to the chemoresistant breast cancer cells so that the drug-enhanced cytotoxicity to cancer cells increases with a minimal toxicity to the non-cancerous cells? We assume that a targeted delivery system is an utmost requirement whether it is delivering siRNA to silence chemoresistant genes or an actual chemodrug which will kill cancer cells without killing non- cancerous cells. To address the chemoresistance as well as off-target toxicity, a targeted delivery system for doxorubicin needs to be developed which should be innovative, comparable and can minimize the toxicity to other non-cancerous cells. And (2) a strategy needs to be in place to determine whether the targeted nanoparticles will carry both doxorubicin and siRNA within the same particles or in different particles to get the best results preventing chemoresistance and limiting off- target toxicity. Our hypothesis is that delivering doxorubicin and MDR-silencing siRNAs separately by targeted nanoparticle system will enhance the cellular toxicity and antitumor effects as compared to a targeted nanoparticle system that delivers the drug and siRNA simultaneously. This hypothesis will be tested through two specific aims: Aim 1: Targeted delivery of doxorubicin liposomes for Her-2 positive breast cancer treatment. Aim 2: Assess whether the targeted nanoparticles will carry both doxorubicin and siRNA within the same particles or in different particles to get the best results preventing chemoresistance and limiting off-target toxicity.