Harnessing the biophysics of multivalent nanoparticle adhesion to control cell targeting and internalization

摘要 纳米载体造影剂和药物递送剂的靶向递送具有令人兴奋的潜力 用于治疗人类重大疾病，但需要新的战略来最大限度地提高靶向效率 和选择性。纳米粒子的一个强大属性是能够与靶形成多个键 细胞，从而提高整体黏附强度和内化率。但是，我们目前 对在分子水平上控制多价纳米颗粒结合的因素知之甚少。 解决这一限制将极大地影响定向交付领域，并使 对多价纳米颗粒粘附性的前所未有的控制。最大的挑战之一是 控制表达靶分子的正常细胞和疾病细胞之间的靶向选择性 在不同的层面上。理想情况下，纳米颗粒将表现出超选择性，这样就可以像开关一样 在正常细胞和病变细胞之间观察到结合效率的变化。到目前为止， 超选择性只在计算模型中观察到，但实验证明 仍然是目标领域的一个主要目标。在以前的工作中，我们开发了新颖的实验 评价多价纳米粒子黏附动力学的方法和计算模拟 所谓的纳米粘合动力学(NAD)，我们用它来揭示有关键的新信息 数量、动态和力量。在这个提案中，我们将把我们的实验和模拟 工具集成到一个通用且强大的设计平台，可用于控制纳米颗粒结合 到活细胞，并在活细胞内内化。我们将使用血管炎症，特别是靶子 ICAM-1作为这项工作的模式系统，由于我们过去的经验，有大量的亲和力 已发表文献中的分子，以及与重大疾病的联系。此外，以前的工作 已经确定了对ICAM-1进行超选择性靶向的必要性。我们将首先添加新的 对NAD模拟的能力，包括纳入纳米颗粒的初始附着 从自由溶液和方法的扩展到纳米棒。第二阶段将重点放在 测试分子键性能，使用不同的流动室进行新的实验 具有不同键性质的抗ICAM-1黏附分子面板，以及一类 我们假设的有弹性的多肽连接物将作为分子弹簧来减少机械 力量。该项目的最后阶段将侧重于使工作适应现场环境 内皮细胞，并使用NAD模拟来设计和测试预期的亲和力分子- 对正常和炎症表现出超选择性靶向行为的纳米颗粒制剂 内皮细胞。具体目标包括：(1)将NAD仿真框架应用于初始建模 附着体和纳米棒，(2)评估新的分子键性质，(3)评估多价 与内皮细胞的黏附，以及(4)设计一种具有超选择性的纳米载体。在 工作结束后，我们将处于理想的位置来设计具有独特性能的纳米载体 针对不同疾病的粘附性，以模拟工具为关键。 这将使我们能够超越直接的概念，如专用性和热力学/ 亲和力，而不是为不同的疾病情景量身定做粘附性，最终实现 超选择性等行为。获得这种能力完全是通过猜测下的实验获得的 而支票格式在时间、金钱和精力方面都过于昂贵。此外， 模拟设计工具将提供解决以下限制和约束所需的多功能性 在活体条件下会遇到。未来的工作将寻求验证我们新的定向交付 概念使用炎症、动脉粥样硬化、缺血再灌注损伤的体内动物模型， 和癌症。

ABSTRACT Targeted delivery of nanocarrier contrast and drug delivery agents holds exciting potential for treating major human diseases, but new strategies are needed to maximize targeting efficiency and selectivity. A powerful attribute of nanoparticles is the ability to form multiple bonds with target cells, thereby enhancing overall adhesion strength and internalization rate. However, we currently know little about the factors that govern multivalent nanoparticle binding at the molecular level. Addressing this limitation would dramatically impact the field of targeted delivery and enable unprecedented control over multivalent nanoparticle adhesion. One of the biggest challenges is controlling targeting selectivity between normal and diseased cells that express the target molecule at different levels. Ideally the nanoparticle would display superselectivity, such that a switch-like change in binding efficiency is observed between normal and diseased cells. To date, superselectivity has only been observed in a computational model, but experimental demonstration remains a major goal in the targeting field. In previous work, we developed novel experimental methods for assessing multivalent nanoparticle adhesion dynamics and a computational simulation called Nano Adhesive Dynamics (NAD) that we used to uncover new information about bond number, dynamics, and forces. In this proposal, we will transform our experimental and simulation tools into a versatile and robust design platform that could be used to control nanoparticle binding to, and internalization within, live cells. We will use vascular inflammation, specifically the target ICAM-1, as a model system for this work due to our past experience, large inventory of affinity molecules in published literature, and connection to major diseases. Furthermore, previous work has already established the need for superselective targeting of ICAM-1. We will first add new capabilities to NAD simulations, including incorporation of the initial attachment of nanoparticles from free solution and extension of the methods to nanorods. The second phase will focus on testing molecular bond properties, with new flow chamber experiments performed using a diverse panel of anti-ICAM-1 adhesion molecules with different bond properties, as well as a class of springy peptide linkers that we hypothesize will act as molecular springs that reduce mechanical forces. The final phase of the project will be focused on adapting the work to the context of live endothelial cells, and using the NAD simulations to design and test prospective affinity molecule- nanoparticle formulations that exhibit superselective targeting behavior to normal and inflamed endothelium. The Specific Aims include: (1) advance the NAD simulation framework to model initial attachment and nanorods, (2) evaluate new molecular bond properties, (3) assess multivalent adhesion to endothelial cells, and (4) design a nanocarrier that displays superselectivity. At the conclusion of the work, we will be in ideal position to design nanocarriers that possess unique adhesive properties for targeting different diseases, with the simulation tool serving as the linchpin. This will allow us to go beyond straightforward concepts such as specificity and thermodynamics/ avidity, and instead tailor adhesion for different disease scenarios and ultimately achieve advanced behavior such as superselectivity. Obtaining this capability entirely from experiments under a guess and check format would be far too costly in terms of time, money, and effort. Furthermore, the simulation design tool will offer the versatility needed to address limitations and constrains that will be encountered under in vivo conditions. Future work will seek to validate our new targeted delivery concepts using in vivo animal models of inflammation, atherosclerosis, ischemia-reperfusion injury, and cancer.