Epshtein Mark, Gounis Matthew J, Bogdanov Alexei A
Department of Radiology, UMASS Chan Medical School, Worcester, Massachusetts 01655, United States.
New England Center for Stroke Research, UMASS Chan Medical School, Worcester, Massachusetts 01655, United States.
ACS Omega. 2024 Jun 24;9(27):29391-29400. doi: 10.1021/acsomega.4c01525. eCollection 2024 Jul 9.
X-ray attenuating contrast agents for imaging thrombi directly during endovascular thrombectomy (EVT) are urgently needed for shortening the wait time for treatment and for reducing the chances of blood clot fragmentation. Neutrophil extracellular traps (NETs) are a product of an innate immune system response by which neutrophils release decondensed chromatin strands decorated with granule and cytosolic proteins, including neutrophil elastase and citrullinated histone H3 (CitH3). NETs are frequently found within fibrous thrombi in pathology and represent a promising target for thrombi-specific imaging agents due to their common occurrence in human cerebrovascular thrombi. We designed and tested 200 nm lipid vesicles (LV) formulated in the presence of a combination of hydrophilic and hydrophobic computed tomography (CT) contrast agents with resultant efficacy of X-ray attenuation corresponding to 312 ± 54 mg/mL iodine. The LV incorporated -cyclooctene-terminated pegylated distearoylphosphatidylethanolamine (TCO-PEG-DSPE) for rapid conjugation of methyltetrazine(mTz)-modified monoclonal immunoglobulin G (IgG) with anti-citH3 binding specificity. By using differential fluorescent labeling of the antibody and lipid components, we determined that 80 ± 3% of mTz-linked IgG coprecipitated with the LV after conjugation in contrast to 0.1-0.2% of control IgG. The engineered NET-specific LV were tested in vitro using differentiated human HL60 promyeloblasts (dHL60) as a standard model of NETing neutrophils. Using fibrin meshwork-incorporated dHL60 as well as monolayer cell cultures, we determined that anti-citH3 LV showed specific and high-affinity binding to dHL60 cells, which were stimulated to undergo NETosis. This work suggests the high promise of NET-specific agents in providing thrombus-specific imaging contrast during EVT.
在血管内血栓切除术（EVT）期间直接成像血栓的X射线衰减造影剂对于缩短治疗等待时间和减少血凝块破碎的几率至关重要。中性粒细胞胞外陷阱（NETs）是先天免疫系统反应的产物，中性粒细胞通过该反应释放出装饰有颗粒和胞质蛋白（包括中性粒细胞弹性蛋白酶和瓜氨酸化组蛋白H3（CitH3））的解聚染色质链。NETs在病理学中经常出现在纤维血栓内，由于它们在人类脑血管血栓中普遍存在，因此是血栓特异性成像剂的一个有前景的靶点。我们设计并测试了200 nm脂质体（LV），其在亲水性和疏水性计算机断层扫描（CT）造影剂组合存在的情况下配制，X射线衰减效果相当于312±54 mg/mL碘。LV掺入了环辛烯封端的聚乙二醇化二硬脂酰磷脂酰乙醇胺（TCO-PEG-DSPE），用于与具有抗CitH3结合特异性的甲基四嗪（mTz）修饰的单克隆免疫球蛋白G（IgG）快速偶联。通过对抗体和脂质成分进行差异荧光标记，我们确定与对照IgG的0.1-0.2%相比，80±3%的mTz连接的IgG在偶联后与LV共沉淀。使用分化的人HL60早幼粒细胞（dHL60）作为NET形成中性粒细胞的标准模型，对工程化的NET特异性LV进行了体外测试。使用掺入纤维蛋白网络的dHL60以及单层细胞培养物，我们确定抗CitH3 LV对被刺激发生NETosis的dHL细胞表现出特异性和高亲和力结合。这项工作表明NET特异性试剂在EVT期间提供血栓特异性成像造影方面具有很高的前景。
