Gemcitabine lipid prodrug nanoparticles: Switching the lipid moiety and changing the fate in the bloodstream

Abstract

A simple approach to achieve a lipoprotein (LP)–mediated drug delivery is to trigger the spontaneous drug insertion into endogenous lipoproteins in the bloodstream, by means of its chemical modification. Nanoparticles (NPs) made of the squalene–gemcitabine (SQGem) conjugate were found to have a high affinity for plasma lipoproteins while free gemcitabine did not, suggesting a key role of the lipid moiety in this event. Whether the drug conjugation to cholesterol, one of the major lipoprotein-transported lipids, could also promote an analogous interaction was a matter of question. NPs made of the cholesterol–gemcitabine conjugate (CholGem) have been herein thoroughly investigated for their blood distribution profile both in vitro and in vivo. Unexpectedly, contrarily to SQGem, no trace of the CholGem prodrug could be found in the lipoprotein fractions, nor was it interacting with albumin. The investigation of isolated NPs and NPs/LPs physical mixtures provided a further insight into the lack of interaction of CholGem NPs with LPs. Although essential for allowing the self–assembly of the prodrug into nanoparticles, the lipid moiety may not be sufficient to elicit interaction of the conjugated drug with plasma lipoproteins but the whole NP physicochemical features must be carefully considered.

Introduction

The ability of lipoproteins (LPs) to transport drugs has been described as early as 1981 (Counsell and Pohland, 1982, Firestone et al., 1984, Gal et al., 1981, Masquelier et al., 1986). Unlike most synthetic nanocarriers, LPs provide biocompatibility, biodegradability and non-immunogenicity (Thaxton et al., 2016). In addition, their structure makes them suitable for drug delivery thanks to: (i) a small size ranging from 5 nm (high–density lipoproteins (HDL)) to 100 nm (very low–density lipoproteins (VLDL)) preventing rapid clearance by the cells of the reticuloendothelial system, (ii) a hydrophobic core that could transport hydrophobic drugs, and (iii) a more hydrophilic outer envelope for amphiphilic molecules loading (Davis and Vance, 1996).

On the other hand, lipoproteins are attractive drug carriers to treat, at least theoretically, any pathological situation that implies their tissue accumulation, in particular neoplastic diseases. Indeed, due to higher cholesterol and cholesteryl esters requirements compared to healthy tissues, most tumors overexpress the low–density lipoprotein (LDL) receptor (LDLR) (Firestone, 1994, Vitols, 1991) and/or the HDL scavenger receptor type B1 (SR–B1) (Mooberry et al., 2016). Accordingly, along with the fact that VLDL and chylomicrons suffer from a low plasma half–life (Thaxton et al., 2016), LDL and HDL have been the predominant lipoproteins investigated for selective drug delivery to tumors (Mahmoudian et al., 2018). Applications in oncology have been largely documented in the past years, but similar strategies have been pursued to target other LP–accumulating tissues such as atherosclerotic plaques (Chen et al., 2020a, Tabas et al., 2007) or organs such as the brain (Chen et al., 2020b, Dehouck et al., 1997).

In this context, the in vitro loading of isolated lipoproteins with various drug molecules has been proposed. It includes conjugation of the drug to the apolipoprotein moieties, its insertion in the phospholipid outer layers or its encapsulation into the hydrophobic core of the LP (Zhu and Xia, 2017). Lipoproteins can be obtained from fresh donor plasma through a variety of separation techniques according to their size, density or apolipoprotein content (e.g., ultracentrifugation, gel permeation, size exclusion chromatography, affinity chromatography, immunocapture assays) (Busbee et al., 1981, Havel et al., 1955, Tadey and Purdy, 1995, Wasan et al., 1999, Watanabe et al., 2012). However, the need to isolate LPs from fresh plasma is a major burden for their use in routine clinical practice. All these methods are indeed low yielding and time consuming, not to mention variations between plasma donors making batch reproducibility difficult. The inherent risk they pose in terms of blood contamination and LP denaturation during the disruptive extraction and loading processes represents another concern with this approach (Busatto et al., 2020).

Formulation of lipoprotein–like carriers has emerged as an alternative strategy. It relies on the in vitro assembly of commercially available lipids in proportions close to that found in natural lipoproteins, therefore bypassing the LP–harvesting step (Corbin and Zheng, 2007, Huang et al., 2015, Pittman et al., 1987). Going beyond simple mimicking, surface–functionalization of these artificial LPs with various ligands has even made it possible to reroute them from their natural pathways and promote their accumulation in target tissues overexpressing other specific receptors (Chen et al., 2007, Corbin et al., 2013, Han et al., 2020, Meng et al., 2015, Zhang et al., 2010, Zheng et al., 2005). Although promising, this approach remains quite laborious since it requires the opportune formulation of a variety of lipids (i.e., phospholipids, cholesterol, cholesterol esters, triglycerides) and apolipoproteins. It also suffers from the fact that these apolipoproteins have to be extracted from human serum, and are difficult to handle because of their poor water-solubility and large size (Corbin and Zheng, 2007).

Overall, the complex production and storage of both the plasma–derived and the artificial lipoproteins, along with the aforementioned concerns, have hindered any industrial transposition and clinical application of LP-based drug delivery systems to date.

It is therefore evident that being able to trigger a drug–lipoprotein interaction directly in the blood circulation would be an easier way to exploit the natural transport capacity of lipoproteins.

We have recently discovered that the conjugation of gemcitabine, a hydrophilic anticancer drug, to the lipophilic squalene chain resulted in a preferential accumulation of the prodrug into lipoproteins, while the free drug did not show any affinity for the latter (Sobot et al., 2017a). Following this interaction, the LPs acted as indirect carriers transporting the squalene-gemcitabine (SQGem) prodrug toward high LP–accumulating cancer cells, leading to a higher therapeutic efficacy compared to free gemcitabine (Sobot et al., 2017b).

As lipoproteins are the natural transporters of cholesterol in the blood circulation, the question remains whether the conjugation of gemcitabine (as a model drug) to cholesterol could provide an even better interaction with endogenous lipoproteins.

Therefore, in the present study, we have explored the fate of nanoparticles (NPs) made of the cholesterol–gemcitabine (CholGem) conjugate both in vitro after incubation with human blood and in vivo after intravenous administration to rodents. Further information was then obtained by directly analyzing the interactions of these NPs with isolated blood components (i.e., LDL, HDL, albumin). The conjugation to the cholesterol resulted in the formation of a prodrug and the activity of the gemcitabine is recovered only after its release from the conjugate following the in vivo enzymatic cleavage of the carbamate bond (Ghosh and Brindisi, 2015, Rautio et al., 2008). However, the assessment of a potential therapeutic activity of the CholGem NPs was not within the scope of this work, which was voluntarily focused on the evaluation of interactions with the components of biological media.

Altogether, these results contribute to shed light on what could drive the in vivo fate of NPs made of lipid prodrugs.