Albumin-driven disassembly of lipidic nanoparticles: the specific case of the squalene-adenosine nanodrug.

In the field of nanomedicine, nanostructured nanoparticles (NPs) made of self-assembling prodrugs emerged in the recent years with promising properties. In particular, squalene-based drug nanoparticles have already shown their efficiency through in vivo experiments. However, a complete pattern of their stability and interactions in the blood stream is still lacking. In this work we assess the behavior of squalene-adenosine (SQAd) nanoparticles - whose neuroprotective effect has already been demonstrated in murine models - in the presence of fetal bovine serum (FBS) and of bovine serum albumin (BSA), the main protein of blood plasma. Extensive physicochemical characterizations were performed using Small Angle Neutron Scattering (SANS), cryogenic transmission electron microscopy (Cryo-TEM), circular dichroism (CD), steady-state fluorescence spectroscopy (SSFS) and isothermal titration calorimetry (ITC) as well as in silico by means of ensemble docking simulations with human serum albumin (HSA). Significant changes in the colloidal stability of the nanoparticles in the presence of serum albumin were observed. SANS, CD and SSFS analyses demonstrated an interaction between SQAd and BSA, with a partial disassembly of the nanoparticles in the presence of BSA and the formation of a complex between SQAd and BSA. The interaction free energy of SQAd nanoparticles with BSA derived from ITC experiments, is about -8 kcal mol-1 which is further supported in silico by ensemble docking simulations. Overall, our results show that serum albumin partially disassembles SQAd nanoparticles by extracting individual SQAd monomers from them. As a consequence, the SQAd nanoparticles would act as a circulating reservoir in the blood stream. The approach developed in this study could be extended to other soft organic nanoparticles.

Translation of nanomedicines from lab to industrial scale synthesis: The case of squalene-adenosine nanoparticles.

A large variety of nanoparticle-based delivery systems have become increasingly important for diagnostic and/or therapeutic applications. Yet, the numerous physical and chemical parameters that influence both the biological and colloidal properties of nanoparticles remain poorly understood. This complicates the ability to reliably produce and deliver well-defined nanocarriers which often leads to inconsistencies, conflicts in the published literature and, ultimately, poor translation to the clinics. A critical issue lies in the challenge of scaling-up nanomaterial synthesis and formulation from the lab to industrial scale while maintaining control over their diverse properties. Studying these phenomena early on in the development of a therapeutic agent often requires partnerships between the public and private sectors which are hard to establish. In this study, through the particular case of squalene-adenosine nanoparticles, we reported on the challenges encountered in the process of scaling-up nanomedicines synthesis. Here, squalene (the carrier) was functionalized and conjugated to adenosine (the active drug moiety) at an industrial scale in order to obtain large quantities of biocompatible and biodegradable nanoparticles. After assessing nanoparticle batch-to-batch consistency, we demonstrated that the presence of squalene analogs resulting from industrial scale-up may influence several features such as size, surface charge, protein adsorption, cytotoxicity and crystal structure. These analogs were isolated, characterized by multiple stage mass spectrometry, and their influence on nanoparticle properties further evaluated. We showed that slight variations in the chemical profile of the nanocarrier's constitutive material can have a tremendous impact on the reproducibility of nanoparticle properties. In a context where several generics of approved nanoformulated drugs are set to enter the market in the coming years, characterizing and solving these issues is an important step in the pharmaceutical development of nanomedicines.

Adenosine and lipids: A forced marriage or a love match?

Adenosine is a fascinating compound, crucial in many biochemical processes: this ubiquitous nucleoside serves as an essential building block of RNA, is also a component of ATP and regulates numerous pathophysiological mechanisms via binding to four extracellular receptors. Due to its hydrophilic nature, it belongs to a different world than lipids, and has no affinity for them. Since the 1970's, however, new discoveries have emerged and prompted the scientific community to associate adenosine with the lipid family, especially via liposomal preparations and bioconjugation. This seems to be an arranged marriage, but could it turn into a true love match? This review considered all types of unions established between adenosine and lipids. Even though exciting supramolecular structures were observed with adenosine-lipid conjugates, as well as with liposomal preparations which resulted in promising pre-clinical results, the translation of these technologies to the clinic is still limited.

Towards a clinical application of freeze-dried squalene-based nanomedicines.

Squalene-adenosine (SQAd) nanoparticles (NPs) were found to display promising pharmacological activity similar to many other nanomedicines, but their long-term stability was still limited, and their preparation required specific know-how and material. These drawbacks represented important restrictions for their potential use in the clinic. Freeze-drying nanoparticles is commonly presented as a solution to allow colloidal stability, but this process needs to be adapted to each nanoformulation. Hence, we aimed at developing a specific protocol for freeze-drying SQAd NPs while preserving their structural features. NPs were lyophilised, resuspended and analysed by dynamic light scattering, atomic force microscopy and small-angle scattering. Among four different cryoprotectants, trehalose was found to be the most efficient in preserving NPs physico-chemical characteristics. Interestingly, we identified residual ethanol in NP suspensions as a key parameter which could severely affect the freeze-drying outcome, leading to NPs aggregation. Long-term stability was also assessed. No significant change in size distribution or zeta potential could be detected after three-month storage at 4 °C. Finally, freeze-dried NPs innocuity was checked in vitro on cultured hepatocytes and in vivo on mice. In conclusion, optimisation of freeze-drying conditions resulted in safe lyophilised SQAd NPs that can be easily stored, shipped and simply reconstituted into an injectable form.

Squalene-Adenosine Nanoparticles: Ligands of Adenosine Receptors or Adenosine Prodrug?

Adenosine receptors (ARs) represent key drug targets in many human pathologies, including cardiovascular, neurologic, and inflammatory diseases. To overcome the very rapid metabolization of adenosine, metabolically stable AR 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 (SQAd) nanoparticles (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: 1) SQAd bioconjugates, which constitute the NP building blocks, act directly as AR ligands; or 2) 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 100 µM 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 lipoprotein 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 the AR pathway, evidenced by fluctuations of the second messenger cAMP. This deeper understanding of the SQAd NPs mechanism of action provides a strong rational for extending the pharmaceutical use of this nanoformulation.

Circulating Lipoproteins: A Trojan Horse Guiding Squalenoylated Drugs to LDL-Accumulating Cancer Cells.

Selective delivery of anticancer drugs to rapidly growing cancer cells can be achieved by taking advantage of their high receptor-mediated uptake of low-density lipoproteins (LDLs). Indeed, we have recently discovered that nanoparticles made of the squalene derivative of the anticancer agent gemcitabine (SQGem) strongly interacted with the LDLs in the human blood. In the present study, we showed both in vitro and in vivo that such interaction led to the preferential accumulation of SQGem in cancer cells (MDA-MB-231) with high LDL receptor expression. As a result, an improved pharmacological activity has been observed in MDA-MB-231 tumor-bearing mice, an experimental model with a low sensitivity to gemcitabine. Accordingly, we proved that the use of squalene moieties not only induced the gemcitabine insertion into lipoproteins, but that it could also be exploited to indirectly target cancer cells in vivo.