Squalene-adenosine nanoparticles: ligands of adenosine receptors or adenosine prodrug?

Adenosine receptors (ARs) represent key drug targets in many human pathologies, including cardiovascular, neurological and inflammatory diseases. To overcome the very rapid metabolization of adenosine, metabolically stable ARs agonists and antagonists were developed. However, few of these molecules have reached the market due to efficacy and safety issues. Conjugation of adenosine to squalene to form squalene-adenosine nanoparticles (SQAd NPs) dramatically improved the pharmacological efficacy of adenosine, especially for neuroprotection in stroke and spinal cord injury. However, the mechanism by which SQAd NPs displayed therapeutic activity remained totally unknown. In the present study, two hypotheses were discussed: (i) either SQAd bioconjugates, which constitute the NPs building blocks, act directly as ARs ligands, or (ii) adenosine, once released from intracellularly processed SQAd NPs, interacts with these receptors. The first hypothesis was rejected, using radioligand displacement assays, as no binding to human ARs was detected up to 1 μM of SQAd, in the presence of plasma. Hence, the second hypothesis was examined. SQAd NPs uptake by HepG2 cells, which was followed using radioactive and fluorescence tagging, was found to be independent of equilibrative nucleoside transporters but rather mediated by low-density lipoproteins receptors. Interestingly, it was observed that after cell internalization, SQAd NPs operated as an intracellular reservoir of adenosine, followed by a sustained release of the nucleoside in the extracellular medium. This resulted in a final paracrine-like activation of ARs pathway, evidenced by fluctuations of the second messenger cAMP. This deeper understanding of SQAd NPs mechanism of action provides now strong rational for extending the pharmaceutical use of this nanoformulation.