The alternative complement pathway and hemocompatibility of nanosurfaces

 描述(由申请人提供)：尽管作为一种治疗工具具有巨大的潜力，但工程纳米颗粒的临床使用也与严重的安全问题有关。全身注射后，纳米颗粒与血液蛋白质相互作用，导致危及生命的超敏反应。免疫细胞摄取含有抗癌毒素的纳米制剂会导致严重的免疫抑制和剂量限制毒性。补体级联的激活是导致工程纳米材料的许多副作用和免疫摄取的原因。在初步数据中，我们证明通过替代途径激活补体是中性粒细胞、单核细胞、淋巴细胞和血小板摄取氧化铁纳米颗粒的主要原因。尽管事实表明，在许多类型的纳米制剂中，替代途径对于补体激活是必不可少的，但减轻替代途径对纳米颗粒激活的策略实际上是不存在的。这一建议的新贡献是开发基于天然抑制蛋白的纳米表面偶联补体抑制剂。这些蛋白质已被用于补体相关疾病的治疗，但从未被评估为保护纳米表面免受补体的影响。我们的初步数据有力地支持了这一假设，即天然替代途径抑制剂的结合将显著改善纳米粒的血液相容性。我们确定了以下具体目标：1)在硅胶中设计替代途径抑制剂，用于随后与纳米表面的偶联。我们将对补体因子和纳米颗粒表面的抑制蛋白进行三维计算机模拟，以确定候选抑制剂和偶联策略；2)确定表面偶联抑制剂的补体抑制效率。我们将过度表达抑制蛋白，或者化学合成更小的多肽。这些抑制剂将通过工程半胱氨酸基连接到各种类型的纳米颗粒上。我们将确定结合抑制剂的效率作为抑制剂密度、Linke类型、纳米颗粒大小和表面化学(电荷、靶向抗体和荧光染料的存在)的函数。这些实验将确定最有效的抑制剂和偶联策略；3)确定抑制剂在改善药物传递纳米平台的血液相容性方面的效率。我们将准备载满化疗药物的纳米平台。这些纳米粒子将被抑制剂修饰，并使用健康人和癌症患者的血液来测试补体激活和免疫细胞摄取。这一结果将对指导未来抑制剂修饰纳米平台的临床前和临床开发具有重要意义。

 DESCRIPTION (provided by applicant): While having tremendous potential as a therapeutic tool, clinical use of engineered nanoparticles has also been associated with serious safety concerns. Following systemic injection, nanoparticles interact with blood proteins causing life-threating hypersensitivity. The uptake of nanoformulations loaded with anticancer toxins by immune cells causes' severe immunosuppression and dose-limiting toxicity. Activation of complement cascade is responsible for many side effects and immune uptake of engineered nanomaterials. In the preliminary data, we demonstrate that activation of complement via the alternative pathway is responsible for the majority of uptake of iron oxide nanoparticles by neutrophils, monocytes, lymphocytes, and platelets. Despite the fact that the alternative pathway has been shown to be essential for complement activation in many types of nanoformulations, the strategies to mitigate the alternative pathway activation on nanoparticles are virtually non-existent. The novel contribution of this proposal is to develop nanosurface-conjugated complement inhibitors based on natural inhibitor proteins. These proteins have been used in the therapeutics of complement-related disorders but have never been evaluated for protecting nanosurfaces against complement. Our preliminary data strongly support the hypothesis that conjugation of the natural alternative pathway inhibitors will significantly improv hemocompatibility of nanoparticles. We established the following Specific Aims: 1) Design alternative pathway inhibitors in silico for subsequent conjugation to nanosurfaces. We will perform 3-D computer modeling of the complement factors and the inhibitor proteins on nanoparticle surface to identify candidate inhibitors and conjugation strategies; 2) determine the complement inhibition efficiency of surface conjugated inhibitors. We will overexpress the inhibitor proteins, or chemically synthesize smaller polypeptides. The inhibitors will be conjugated to various types of nanoparticles via an engineered cysteine group. We will determine the efficiency of the conjugated inhibitors as a function of the inhibitor density, linke type, nanoparticle size, and surface chemistry (charge, presence of targeting antibody and fluorescent dye). These experiments will determine the most efficient inhibitors and conjugation strategies; 3) determine the efficiency of the inhibitors in improving hemocompatibility of drug delivery nanoplatforms. We will prepare nanoplatforms loaded with chemotherapy drugs. The nanoparticles will be modified with inhibitors and tested for complement activation and immune cell uptake using blood from healthy individuals and cancer patients. The results will be highly beneficial in guiding future preclinical and clinical development of inhibitor-decorated nanoplatforms.