In Vivo FRET Imaging to Predict the Risk Associated with Hepatic Accumulation of Squalene-Based Prodrug Nanoparticles.

Förster resonance energy transfer (FRET) is used here for the first time to monitor the in vivo fate of nanoparticles made of the squalene-gemcitabine prodrug and two novel derivatives of squalene with the cyanine dyes 5.5 and 7.5, which behave as efficient FRET pair in the NIR region. Following intravenous administration, nanoparticles initially accumulate in the liver, then they show loss of their integrity within 2 h and clearance of the squalene bioconjugates is observed within 24 h. Such awareness is a key prerequisite before introduction into clinical settings.

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.

Conjugation of squalene to gemcitabine as unique approach exploiting endogenous lipoproteins for drug delivery.

Once introduced in the organism, the interaction of nanoparticles with various biomolecules strongly impacts their fate. Here we show that nanoparticles made of the squalene derivative of gemcitabine (SQGem) interact with lipoproteins (LPs), indirectly enabling the targeting of cancer cells with high LP receptors expression. In vitro and in vivo experiments reveal preeminent affinity of the squalene-gemcitabine bioconjugates towards LP particles with the highest cholesterol content and in silico simulations further display their incorporation into the hydrophobic core of LPs. To the best of our knowledge, the use of squalene to induce drug insertion into LPs for indirect cancer cell targeting is a novel concept in drug delivery. Interestingly, not only SQGem but also other squalene derivatives interact similarly with lipoproteins while such interaction is not observed with liposomes. The conjugation to squalene represents a versatile platform that would enable efficient drug delivery by simply exploiting endogenous lipoproteins.

Synthesis of a deuterated probe for the confocal Raman microscopy imaging of squalenoyl nanomedicines.

The synthesis of ω-di-(trideuteromethyl)-trisnorsqualenic acid has been achieved from natural squalene. The synthesis features the use of a Shapiro reaction of acetone-d 6 trisylhydrazone as a key step to implement the terminal isopropylidene-d 6 moiety. The obtained squalenic acid-d 6 has been coupled to gemcitabine to provide the deuterated analogue of squalenoyl gemcitabine, a powerful anticancer agent endowed with self-assembling properties. The Raman spectra of both deuterated and non-deuterated squalenoyl gemcitabine nanoparticles displayed significant Raman scattering signals. They revealed no differences except from the deuterium peak patterns in the silent spectral region of cells. This paves the way for label-free intracellular trafficking studies of squalenoyl nanomedicines.

In vitro investigation of multidrug nanoparticles for combined therapy with gemcitabine and a tyrosine kinase inhibitor: Together is not better.

Combined therapy with gemcitabine and tyrosine-kinase inhibitors (i.e., sunitinib) has already demonstrated important benefits in pancreatic cancer treatment. Further therapeutic advantage could be achieved by their co-loading in a single nanoscale system, which enables (i) the co-existence of drugs with different mechanisms of action and pharmacokinetic profiles and (ii) the fine tuning of their release rate overcoming the rapid clearance often observed with free drugs. In this context, the already validated squalenoylation approach has been applied to the design of a multidrug nanoparticle (NP) made by co-self-assembly of the squalene-based prodrugs of gemcitabine (SQGem) and sunitinib (SQSun). We hypothesized that co-delivering of SQGem and SQSun in a single nanoparticle was capable to increase their cytotoxicity on MIA PaCa-2 pancreatic cancer cells compared to the monodrug NPs. Nevertheless, multidrug NPs (i.e., SQGem/SQSun NPs) were as efficient as the physical mixture of the individual monodrug NPs (SQGem NPs + SQSun NPs) thus suggesting that the cytotoxicity raised from the exposure of the cells simultaneously to the two bioconjugates rather than to their original loading into a single or two different nanoparticles. To be noted that the lack of differences in static 2D cultures does not exclude a different behavior in dynamic conditions in vivo.

How can nanomedicines overcome cellular-based anticancer drug resistance?

Despite the great progress in the field of cancer research, complete remission of cancer patients is a rarely seen outcome in clinics. The failure of a plethora of promising anticancer drugs is mostly due to the insurgence of multidrug resistance (MDR) phenomena with various factors, both physio-pathological and cellular, being responsible for their development. Over the past 40 years, the impressive progress in the nanotechnology field and its application in medicine (i.e., nanomedicine) enabled the development of novel cancer treatments with improved specificity and efficacy, mainly referring to the possibility to overcome MDR. However, the emergence of many nanomedicines defined in the literature as "overcoming the resistance" has resulted in very few of them eventually being approved for clinical application and reaching the marketplace (liposomal formulation of doxorubicin (Myocet®, Caelix®) and paclitaxel nanoparticles (Abraxane®), for instance). The capacity of nanomedicines to overcome physio-pathological-based resistance by enhancing drug accumulation at the tumor site via passive and ligand-mediated targeting has been largely reviewed in the past few years. This review will specifically focus on the cellular mechanisms involved in the MDR, which will be discussed with respect to the cellular site in which their action is exerted (that is, membrane, cytoplasm or nucleus). A complete overview of various nanomedicines designed to bypass or directly inhibit each of these specific resistance mechanisms will be offered.