Kazmierczak Robert, Choe Elizabeth, Sinclair Jared, Eisenstark Abraham
Cancer Research Center, 3501 Berrywood Drive, Columbia, MO, 65201, USA,
Methods Mol Biol. 2015;1225:151-63. doi: 10.1007/978-1-4939-1625-2_11.
Nanoparticle technology is an emerging approach to resolve difficult-to-manage internal diseases. It is highly regarded, in particular, for medical use in treatment of cancer due to the innate ability of certain nanoparticles to accumulate in the porous environment of tumors and to be toxic to cancer cells. However, the therapeutic success of nanoparticles is limited by the technical difficulty of fully penetrating and thus attacking the tumor. Additionally, while nanoparticles possess seeming-specificity due to the unique physiological properties of tumors themselves, it is difficult to tailor the delivery of nanoparticles or drugs in other models, such as use in cardiac disease, to the specific target. Thus, a need for delivery systems that will accurately and precisely bring nanoparticles carrying drug payloads to their intended sites currently exists. Our solution to this engineering challenge is to load such nanoparticles onto a biological "mailman" (a novel, nontoxic, therapeutic strain of Salmonella typhimurium engineered to preferentially and precisely seek out, penetrate, and hinder prostate cancer cells as the biological delivery system) that will deliver the therapeutics to a target site. In this chapter, we describe two methods that establish proof-of-concept for our cargo loading and delivery system by attaching nanoparticles to the Salmonella membrane. The first method (Subheading 1.1) describes association of sucrose-conjugated gold nanoparticles to the surface of Salmonella bacteria. The second method (Subheading 1.2) biotinylates the native Salmonella membrane to attach streptavidin-conjugated fluorophores as example nanoparticle cargo, with an alternative method (expression of membrane bound biotin target sites using autodisplay plasmid vectors) that increases the concentration of biotin on the membrane surface for streptavidin-conjugated nanoparticle attachment. By directly attaching the fluorophores to our bacterial vector through biocompatible, covalent, and stable bonds, the coupling of bacterial and nanoparticle therapeutic approaches should synergistically lead to improved tumor destruction.
纳米颗粒技术是一种新兴的解决难治性内科疾病的方法。它尤其受到高度重视，因为某些纳米颗粒具有在肿瘤的多孔环境中聚集并对癌细胞产生毒性的固有能力，可用于癌症治疗。然而，纳米颗粒的治疗成功受到完全穿透并因此攻击肿瘤的技术难度的限制。此外，虽然由于肿瘤本身独特的生理特性，纳米颗粒具有表面特异性，但很难将纳米颗粒或药物的递送调整到其他模型中，例如用于心脏病治疗时靶向特定目标。因此，目前需要一种能够准确、精确地将携带药物的纳米颗粒输送到预定部位的递送系统。我们应对这一工程挑战的解决方案是将此类纳米颗粒装载到一个生物“邮递员”（一种经过工程改造的新型、无毒的鼠伤寒沙门氏菌治疗菌株，作为生物递送系统，它能够优先且精确地寻找、穿透并抑制前列腺癌细胞）上，该“邮递员”将把治疗剂递送至目标部位。在本章中，我们描述了两种通过将纳米颗粒附着到沙门氏菌膜上来为我们的货物装载和递送系统建立概念验证的方法。第一种方法（1.1小节）描述了蔗糖共轭金纳米颗粒与沙门氏菌细菌表面的结合。第二种方法（1.2小节）将天然沙门氏菌膜生物素化，以附着链霉亲和素共轭荧光团作为示例纳米颗粒货物，还有一种替代方法（使用自展示质粒载体表达膜结合生物素靶位点）可增加膜表面用于链霉亲和素共轭纳米颗粒附着的生物素浓度。通过生物相容性、共价且稳定的键将荧光团直接附着到我们的细菌载体上，细菌和纳米颗粒治疗方法的结合应能协同提高肿瘤破坏效果。
