Hector Francisco Valdovinos

A compact field-deployable microfluidic system has been developed to improve timelines for the rapid analysis of debris in post-detonation nuclear forensics. We used a high-resolution 3D printer to miniaturize typical laboratory-based procedures into a fieldable platform. Microfluidic half-modules were produced for the purification of Pu from excess U, along with a portable alpha chamber for the following isotopic analysis of the Pu stream. A porous PTFE membrane is soaked with a hydrophobic tributyl phosphate (TBP) solution and is placed between two half-modules; separation is performed as a liquid–liquid extraction in an extraction channel across this membrane, where the forward and back-extractions occur within one complete module. Following separation, a 100 μL sampling of the Pu-bearing stream is injected into a small-footprint 3D printed alpha chamber for isotopic assay via alpha spectrometry as part of an online process. In this first demonstration of microfluidic separation coupled with online alpha spectrometry, high extraction yields have been obtained for Pu (98.9 ± 4.0)% and U (97.5 ± 2.5)%. The process uses less than 800 μL of solution with separation chemistry complete within 45 minutes and subsequent alpha spectrometry initiating 25 minutes after separation.

Peripheral Arterial Disease (PAD) is a chronic occlusion of lower-extremity arteries distal to the aortic bifurcation attributable to atherosclerosis. PAD is associated with an elevated risk of morbidity and mortality as well as of deterioration of the quality of life with claudication and chronic leg ischemia being the most frequent complications. Therapeutic angiogenesis is a promising therapeutic strategy that aims to restore the blood flow to the ischemic limb. In this context, assessing the efficacy of pro-angiogenic treatment using a reliable non-invasive imaging technique would greatly benefit the implementation of this therapeutic approach. Herein, we describe the angiogenesis and perfusion recovery characteristics of a mouse model of PAD via in vivo positron emission tomography (PET) imaging of CD146 expression. For that, ischemia was induced by ligation and excision of the right femoral artery of Balb/C mice and confirmed through Laser Doppler imaging. Ischemia-induced angiogenesis was non-invasively monitored and quantified through PET imaging of CD146 expression in the injured leg using a ⁶⁴Cu-labeled anti-CD146 monoclonal antibody, ⁶⁴Cu-NOTA-YY146, at postoperative days 3, 10 and 17. The CD146-specific character of ⁶⁴Cu-NOTA-YY146 was confirmed via a blocking study performed in another cohort at day 10 after surgery. Tracer uptake was correlated with in situ CD146 expression by histological analysis. PET scans results indicated that ⁶⁴Cu-NOTA-YY146 uptake in the ischemic hindlimb was significantly higher, with a maximum uptake of 14.1 ± 2.0 %ID/g at postoperative day 3, compared to the normal contralateral hindlimb, at all time points (maximum uptake of 2.2 ± 0.2 %ID/g). The pre-injection of a blocking dose resulted in a significantly lower tracer uptake in the operated hindlimb on day 10 after surgery, confirming tracer specificity. CD146/CD31 immunofluorescent co-staining showed excellent correlation between the high uptake of the tracer with in situ CD146 expression levels and a marked co-localization of CD146 and CD31 signals. In conclusion, persistent and specific CD146 uptake of the tracer in the ischemic hindlimb was observed, confirming the feasibility of ⁶⁴Cu-NOTA-YY146 as an imaging agent to monitor the progression of angiogenesis and recovery in future PAD research.

CD146 has been identified as an excellent biomarker for lung cancer as its overexpression in solid tumors has been linked to disease progression, invasion, and metastasis. Previously, our group described a positive correlation between ⁶⁴Cu-labeled YY146 uptake and increased expression of CD146 in six human lung cancer cell lines using subcutaneous tumor models. In this study, we investigate a monoclonal antibody called YY146 for immunoPET imaging of CD146 in two intrapulmonary metastasis models of non-small cell lung cancer (NSCLC). The binding and immunoreactivity of the tracer were assessed by in vitro assays. Radiolabeling of YY146 with positron emitting Cu-64 (⁶⁴Cu-NOTA-YY146) enabled PET imaging of intrapulmonary metastasis. Mice were intravenously injected with two million tumor cells, and CT imaging was used to verify the presence of lung metastases. ⁶⁴Cu-NOTA-YY146 was injected into tumor-bearing mice, and animals were subjected to PET/CT imaging at 4, 24, and 48 h postinjection. Both the average and maximum lung PET signal intensities were quantified and compared between high and low CD146-expressing metastases. Further validation was accomplished through immunofluorescence imaging of resected tissues with CD31 and CD146. In flow cytometry, YY146 revealed strong binding to CD146 in H460 cells due to its high expression with minimal binding to CD146-low expressing H358 cells. Both YY146 and NOTA-YY146 showed similar binding, suggesting that NOTA conjugation did not elicit any negative effects on its binding affinity. Imaging of ⁶⁴Cu-NOTA-YY146 in H460 tumor-bearing mice revealed rapid, persistent, and highly specific tracer accumulation. Uptake of ⁶⁴Cu-NOTA-YY146 in the whole lung was calculated for H460 and H358 as 7.43 ± 0.38 and 3.95 ± 0.47% ID/g at 48 h postinjection (n = 4, p < 0.05), and the maximum lung signals were determined to be 13.85 ± 1.07 (H460) and 6.08 ± 0.73% ID/g (H358) at equivalent time points (n = 4, p < 0.05). To ensure the specificity of the tracer, a nonspecific antibody was injected into H460 tumor-bearing mice. Ex vivo biodistribution and immunofluorescence imaging validated the PET findings. In summary, ⁶⁴Cu-NOTA-YY146 allowed for successful imaging of CD146-expressing intrapulmonary metastases of NSCLC in mice. This preliminary study provides evidence supporting the future clinical utilization of ⁶⁴Cu-NOTA-YY146 for possible treatment monitoring of CD146-targeted therapy or improving patient stratification.