Plasma extracellular vesicles (EVs), especially exosome-like vesicles (ELVs), are being increasingly explored as a source of potential noninvasive disease biomarkers. The discovery of blood-based biomarkers associated with ELVs requires methods that isolate high yields of these EVs without significant contamination with highly abundant plasma proteins and lipoproteins. The rising interest in blood-based EV-associated biomarkers has led to the rapid development of novel EV isolation methods. However, the field suffers from a lack of standardization and often, new techniques are used without critical evaluation. Size exclusion chromatography (SEC) has become the method of choice for rapid isolation of relatively pure EVs from plasma, yet it has technical limitations for certain downstream applications. The recently released exoEasy kit (Qiagen) is a new membrane affinity spin column method for the isolation of highly pure EVs from biofluids with the potential to overcome most of the limitations of SEC. By using multiple complementary techniques we assessed the performance of the exoEasy kit in isolating ELVs from 2 ml of human plasma and compared it with the SEC qEV column (Izon Science). Our data show that exoEasy kit isolates a heterogenous mixture of particles with a larger median diameter, broader size range and a higher yield than the SEC qEV column. The exclusive presence of small RNAs in the particles and the total RNA yield were comparable to the SEC qEV column. Despite being less prone to low density lipoprotein contamination than the SEC qEV column, the overall purity of exoEasy kit EV preparations was suboptimal. The low particle-protein ratio, significant amount of albumin, very low levels of exosome-associated proteins and propensity to triglyceride-rich lipoprotein contamination suggest isolation of mainly non-ELVs and co-isolation of plasma proteins and certain lipoproteins by the exoEasy kit. We demonstrate that performance of exoEasy kit for the isolation of ELVs for biomarker discovery is inferior to the SEC qEV column. This comprehensive evaluation of a novel EV isolation method contributes to the acceleration of the discovery of EV-associated biomarkers and the development of EV-based diagnostics.

The acyclic nucleoside phosphonate (ANP) analogues are broad-spectrum antiviral agents, with potent and selective antiviral activity in vitro and in vivo. The prototype compounds are: (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine (HPMPC, cidofovir), which is active against a wide variety of DNA viruses; 9-(2-phosphonylmethoxyethyl)adenine (PMEA, adefovir), which is active against retro-, herpes- and hepadnaviruses, and (R)-9-(2-phosphonylmethoxypropyl) adenine (PMPA), which is active against retro- and hepadnaviruses. The antiviral action of the ANP analogues is based on a specific interaction of the active diphosphorylated metabolite with the viral DNA polymerase. The long intracellular half-life of the active metabolite accounts for the optimal efficacy in infrequent dosing schedules. The potential of HPMPC as a broad-spectrum anti-DNA virus agent, as originally observed in vitro and in vivo, has been confirmed in clinical trials. HPMPC has recently been commercially released in the USA for the treatment of cytomegalovirus retinitis in AIDS patients. In addition, topical systemic HPMPC is being (or will be) explored for use against other herpesviruses (i.e. herpes simplex virus, Epstein-Barr virus, or varicella-zoster virus), by adenoviruses, or by human papilloma- or polyomaviruses. Intravenous HPMPC is associated with dose-dependent nephrotoxicity, that should be counteracted by prehydration and concomitant administration of probenecid, and by the application of an infrequent dosing schedule. The oral prodrug of PMEA, bis(pivaloyloxymethyl)-PMEA, is currently being evaluated in patients infected with human immunodeficiency virus (HIV) or hepatitis B virus. Finally, preclinical data on the efficacy of PMPA in animal retrovirus models point to its potential usefulness against HIV infections, when given either prophylactically or therapeutically in the treatment of established HIV infections.