Li Junling, Gerlach Rachael L, Jonsson Colleen B, Gray Brian D, Pak Koon Y, Ng Chin K
Department of Radiology, University of Louisville School of Medicine, Louisville, KY.
Center for Predictive Medicine and Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY.
Nucl Med Biol. 2015 Mar;42(3):283-91. doi: 10.1016/j.nucmedbio.2014.11.012. Epub 2014 Dec 5.
Bis(Zn-dipicolylamine (Zn-DPA)) coordination complexes represent a new class of synthetic small molecules that can target anionic phosphatidylserine (PS) in the apoptotic cells with high affinity and specificity. In this study, we labeled Zn-DPA and Cy7-Zn-DPA with different (18)F-prosthetic groups and characterized their uptake in A549 cells infected with influenza A virus from the 2009 pandemic (H1N1pdm).
DPA was labeled with N-succinimidyl 4-(18)F-fluorobenzoate ((18)F-SFB), 4-nitrophenyl 2-(18)F-fluoropropionate ((18)F-NFP), 2-(18)F-Fluoroethyl toslyate ((18)F-FET), and (18)F-aluminum (Al(18)F), respectively. Cy7-DPA was labeled with (18)F-SFB and (18)F-NFP only. The tracers were reconstituted with zinc nitrate before use. Apoptosis in A549 cells was induced by infection with the H1N1pdm virus for 48 h. Three μCi of each tracer was added to each well and incubated at 37 °C. The effect of different prosthetic groups, different MOI, and incubation time on percent cellular uptake was studied. Cell internalization and efflux was evaluated within 2h of incubation. The competitive binding assay was performed with increasing concentration (10(-12)-10(-5)M) of Zn-DPA or Cy7-Zn-DPA prior to the addition of either (18)F-FB-Zn-DPA or (18)F-FB-Cy7-Zn-DPA into each well. IC50 values for the two Zn-DPA analogues were estimated by GraphPad Prism 6.0.
Among all the four prosthetic groups, the (18)F-SFB method provided the highest conjugation yield for DPA and the highest uptake ratio between the infection cells and the control when both Zn-DPA and Cy7-Zn-DPA were present in the complex. The uptake ratio was similar for (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA. Uptake of (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA was proportional to the degree of apoptosis with a plateau at MOI 3. Uptake of (18)F-FB-Cy7-Zn-DPA also increased over incubation time and reached a plateau at 1h, whereas uptake of (18)F-FB-Zn-DPA did not show any significant change over time. Cell internalization studies showed that more than 70% of (18)F-FB-Zn-DPA remained on the cell surface over a time course of 2 hr in the cell media, but over 90% of (18)F-FB-Cy7-Zn-DPA was internalized within 15 min of incubation. IC50 values were estimated to be 1.5±0.3 nM and 26.2±5.1 nM for Zn-DPA and Cy7-Zn-DPA, respectively.
(18)F-SFB was the optimal labeling method for Zn-DPA and Cy7-Zn-DPA with respect to radiochemistry and provided complexes with high target-to-background ratios. (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA appeared to have a completely different internalization mechanism, while Zn-DPA showed higher binding affinity than Cy7-Zn-DPA. Based on these favorable characteristics, (18)F-FB-Zn-DPA and (18)F-FB-Cy7-Zn-DPA should be further evaluated as potential imaging agents for viral infection.
双（锌 - 二吡啶甲胺（Zn - DPA））配位络合物是一类新型合成小分子，能够以高亲和力和特异性靶向凋亡细胞中的阴离子磷脂酰丝氨酸（PS）。在本研究中，我们用不同的(^{18}F) - 连接基团标记了Zn - DPA和Cy7 - Zn - DPA，并对它们在感染2009年大流行甲型流感病毒（H1N1pdm）的A549细胞中的摄取情况进行了表征。
分别用N - 琥珀酰亚胺基4 - （(^{18}F)） - 氟苯甲酸酯（(^{18}F) - SFB）、4 - 硝基苯基2 - （(^{18}F)） - 氟丙酸酯（(^{18}F) - NFP）、2 - （(^{18}F)） - 氟乙基对甲苯磺酸酯（(^{18}F) - FET）和(^{18}F) - 铝（Al(^{18}F)）标记DPA。仅用(^{18}F) - SFB和(^{18}F) - NFP标记Cy7 - DPA。使用前用硝酸锌重构示踪剂。用H1N1pdm病毒感染A549细胞48小时诱导细胞凋亡。向每个孔中加入3μCi的每种示踪剂，并在37℃下孵育。研究了不同连接基团、不同感染复数（MOI）和孵育时间对细胞摄取百分比的影响。在孵育2小时内评估细胞内化和流出情况。在向每个孔中加入(^{18}F) - FB - Zn - DPA或(^{18}F) - FB - Cy7 - Zn - DPA之前，用浓度递增（(10^{-12}) - (10^{-5})M）的Zn - DPA或Cy7 - Zn - DPA进行竞争结合试验。用GraphPad Prism 6.0估计两种Zn - DPA类似物的半数抑制浓度（IC50）值。
在所有四种连接基团中，当络合物中同时存在Zn - DPA和Cy7 - Zn - DPA时，(^{18}F) - SFB方法为DPA提供了最高的偶联产率，以及感染细胞与对照之间的最高摄取率。(^{18}F) - FB - Zn - DPA和(^{18}F) - FB - Cy7 - Zn - DPA的摄取率相似。(^{18}F) - FB - Zn - DPA和(^{18}F) - FB - Cy7 - Zn - DPA的摄取与凋亡程度成正比，在MOI为3时达到平台期。(^{18}F) - FB - Cy7 - Zn - DPA的摄取也随孵育时间增加，并在1小时时达到平台期，而(^{18}F) - FB - Zn - DPA的摄取随时间未显示任何显著变化。细胞内化研究表明，在细胞培养基中2小时的时间进程内，超过70％的(^{18}F) - FB - Zn - DPA保留在细胞表面，但超过90％的(^{18}F) - FB - Cy7 - Zn - DPA在孵育15分钟内被内化。Zn - DPA和Cy7 - Zn - DPA的IC50值估计分别为1.5±0.3 nM和26.2±5.1 nM。
就放射化学而言，(^{18}F) - SFB是Zn - DPA和Cy7 - Zn - DPA的最佳标记方法，并提供了高靶本比的络合物。(^{18}F) - FB - Zn - DPA和(^{18}F) - FB - Cy7 - Zn - DPA似乎具有完全不同的内化机制，而Zn - DPA显示出比Cy7 - Zn - DPA更高的结合亲和力。基于这些有利特性，(^{18}F) - FB - Zn - DPA和(^{18}F) - FB - Cy7 - Zn - DPA应作为病毒感染的潜在成像剂进行进一步评估。
