Development of Anti-Fouling Peptide-Nanoparticle Conjugates for the Delivery of siRNA to Fractures

小干扰RNA(SiRNA)是一类很有前途的药物，但受到递送挑战的限制。裸体 SiRNA在体内的细胞摄取能力差，降解速度快，因此siRNA通常在 纳米颗粒(NP)制剂。绕过降解和摄取不良的细胞外屏障以及 细胞内溶酶体转运，我们的实验室率先开发了pH响应型阳离子双阻断 用于传递siRNA的三元共聚纳米粒(PHCNPs)，大大提高了siRNA的治疗效果 功效。局部体内传递siRNA-pHCNPs已在骨骼和其他组织中显示出希望，但许多 包括许多肌肉骨骼系统在内的疾病需要全身给药。系统地交付 NPS因血清吸附而受阻，导致单核巨噬细胞系统(MPS)摄取不良 循环次数，并可能增强免疫原性。事实上，许多被批准的siRNA-NP疗法 或在发育后期利用MPS在肝脏中积累的优势，针对肝脏疾病。 减少蛋白质-NP的相互作用是改善siRNA-NPs系统递送以达到其他目标的关键 纸巾。纳米粒子的聚乙二醇化修饰(聚乙二醇化)是目前降低蛋白质的标准 吸附，但它也通过阻碍摄取而降低NP的效力，并可能引发免疫反应 由于有抗聚乙二醇单抗。两性离子(ZI)部分在减少蛋白质吸附方面显示出巨大的前景 并且与聚乙二醇组分相比，对NP功能特性的干扰较小。仿生ZI肽(ZIP)是一种 改善聚合物NP药代动力学性质的新途径。特别是，半随机化的 Zips(SrZIP)允许半独立于氨基酸组成的电荷序列测试。这些 总而言之，属性导致假设半随机化ZIP(SrZIP)设计为低 聚集势可用于修饰NPs以改善体循环和siRNA输送 通过减少NP与血清蛋白的相互作用。我们将通过三种方式检验这一假设。在目标1中，我们将 建立srZIP-pHCNP结合物文库，以测试其对血清诱导的聚集的影响 幼稚的pHCNPs和聚乙二醇化的pHCNPs。在Aim 2A中，我们将使用目标细胞在体外测试这些偶联物 (间充质干细胞)和MPS细胞(巨噬细胞)，在Aim 2B中，我们将评估在 PHCNP在体内的循环次数是偶联的。在目标3中，我们将使用已建立的小鼠股骨骨折 研究携带治疗性抗WWP1 siRNA(WW域)的pHCNP骨折堆积的模型 含有E3泛素蛋白Ligase 1，它是骨折愈合的负面调节因子)，我们已经证明 使用局部给药方法加速骨折愈合。在这个项目完成后，我们预计 寻找在聚合物中传递siRNA的新的基于多肽的反聚集方法 纳米粒子，为未来肌肉骨骼应用系统传递siRNA奠定基础 更远一点。

Small interfering RNA (siRNA) are a promising class of drugs that are limited by delivery challenges. Naked siRNA has poor cellular uptake and rapid degradation in vivo, thus siRNA is typically administered in nanoparticle (NP) formulations. To circumvent extracellular barriers of degradation and poor uptake as well as intracellular lysosomal trafficking, our lab pioneered the development of pH-responsive cationic diblock tercopolymer nanoparticles (pHCNPs) for the delivery of siRNA, which greatly improve siRNA therapeutic efficacy. Local in vivo delivery of siRNA-pHCNPs has shown promise in bone and other tissues, but many diseases, including many of the musculoskeletal system require systemic administration. Systemic delivery of NPs is hampered due to serum adsorption, leading to mononuclear phagocyte system (MPS) uptake and poor circulation times, and may potentiate immunogenicity. In fact, many siRNA-NP therapeutics that are approved or in late stages of development take advantage of MPS accumulation in liver, targeting hepatic diseases. Reducing protein-NP interactions is key to improving the systemic delivery of siRNA-NPs to reach other target tissues. Poly(ethylene glycol) (PEG) modification (PEGylation) of NPs is the current standard to reduce protein adsorption, but it also reduces NP efficacy by hampering uptake and may induce immunological responses due to anti-PEG antibodies. Zwitterionic (ZI) moieties have shown great promise in reducing protein adsorption and are less disruptive to NP functional characteristics than PEG. Biomimetic ZI peptides (ZIPs) are a promising new approach to improve polymeric NP pharmacokinetic properties. In particular, semi-randomized ZIPs (srZIPs) allow testing of charge sequence semi-independently of amino acid composition. These attributes altogether lead to the hypothesis that semi-randomized ZIPs (srZIPs) designed with low aggregation potential can be used to modify NPs to improve systemic circulation and siRNA delivery by reducing NP-serum protein interactions. We will test this hypothesis in three ways. In Aim 1, we will generate a library of srZIP-pHCNP conjugates to test the effects on serum-induced aggregation compared to naïve pHCNPs and PEG-pHCNPs. In Aim 2A, we will test these conjugates in vitro using target cells (mesenchymal stem cells) and in MPS cells (macrophages), and in Aim 2B we will evaluate improvements in circulation times of the pHCNP conjugates in vivo. In Aim 3 we will use an established mouse femur fracture model to investigate fracture accumulation of pHCNP bearing therapeutic anti-WWP1 siRNA (WW Domain Containing E3 Ubiquitin Protein Ligase 1, a negative regulator of fracture healing), which we have shown to expedite bone fracture healing in using a local delivery approach. At the completion of this project, we expect to identify new peptide-based anti-aggregation approaches for the delivery of siRNA in polymeric nanoparticles, laying the foundation for future systemic delivery of siRNA for musculoskeletal applications and beyond.