Monoolein-based cubosomes affect lipid profile in HeLa cells
Graphical abstract
Introduction
Nanoparticles (NPs) are a class of functional materials characterized by size-dependent properties, generally defined as engineered structures with at least one dimension less than 100 nm (Kroll et al., 2009), for which applications in medicine as therapeutic drug delivery and/or medical imaging systems have been predicted (Faraji and Wipf, 2009, Kroll et al., 2009). Relevant examples include micelles, liposomes, solid lipid, polymeric, silicon-based, gold, or iron oxide NPs, as well as dendrimers, and quantum dots (Faraji and Wipf, 2009; Panariti el al., 2012). Lipid-based reverse cubic bicontinuous liquid crystalline phases possess a three-dimensional structure consisting of non-intersecting bilayers folded on an infinite periodic minimal surface characterized by a cubic symmetry and organized to form two disconnected, continuous water channels (Hyde, 1989). They were broadly investigated for pharmaceutical purposes in the past as their nanostructure can incorporate molecules of biological relevance (Caboi et al., 2001, Murgia et al., 2001). Remarkably, these peculiar cubic phases can be dispersed in water originating nanoparticles known as cubosomes (Larsson, 1983, Larsson and Tiberg, 2005), often pictured as the non-lamellar counterpart of liposomes. Cubosomes can be easily prepared sterically stabilizing a dispersion of monoolein (MO, Fig. 1A) in water by Pluronic series. By virtue of peculiar characteristics such as high mechanical rigidity, high hydrophobic volume, and the possibility of being biodegraded in vivo through enzyme-catalyzed reactions (carboxylesterases and phosphatases) (Hinton et al., 2014, Mulet et al., 2013), monoolein-based cubosomes were recently proposed for application in theranostic nanomedicine (Caltagirone et al., 2014, Murgia et al., 2013).
Several investigations were conducted on the in vitro cytotoxicity of MO-based nanoparticles, and results were found related to cell line, incubation time, dose, and formulation type (Falchi et al., 2015, Hinton et al., 2014, Murgia et al., 2010, Murgia et al., 2015, Tran et al., 2015). Previous studies also provided evidence that, as result of their internalization, MO-based cubosomes induce accumulation of lipids in treated cells, causing the increase (both in size and number) of the cytoplasmic lipid droplets (LDs) (Caltagirone et al., 2014, Falchi et al., 2015, Murgia et al., 2015). LDs are macromolecular lipid assemblies consisting of neutral lipids, such as triacylglycerols, diacylglycerols, cholesterol esters, and cholesterol, surrounded by a monolayer of phospholipids and associated proteins (Bartz et al., 2007, Khatchadourian and Maysinger, 2009, Przybytkowski et al., 2009, Suzuki et al., 2012). LDs are now regarded as metabolically active organelles, with a particular structure and organization, engaged in a wide range of activities and formed, under physiological conditions, when free (unesterified) fatty acids from exogenous or endogenous sources are available inside the cells (Khatchadourian and Maysinger, 2009, Przybytkowski et al., 2009, Suzuki et al., 2012). Their main functions includes lipid storing and supplying for various cellular needs (β-oxidation, membrane biogenesis, and lipoprotein synthesis) (Suzuki et al., 2012).
Remarkably, although numerous articles were devoted to validate cubosome relevance in nanomedicine, only a small amount of research was undertaken to investigate the impact of these nanoparticles on the cell lipid profile (Falchi et al., 2015). The present investigation aimed to fill this gap by exploring the changes in lipid components occurring in HeLa cells when exposed to short-term treatments with MO-based cubosomes stabilized by Pluronic F108 (MO/PF108). The effect of nanoparticles (at 2 and 4 h of incubation) on HeLa cell viability was preliminary evaluated by the MTT assay. MO/PF108 cubosomes, at non-cytotoxic concentration, were then tested to evaluate their effect on lipid component profile, with particular regard to triacylglycerols (TAG), phospholipids (PL), free cholesterol (FC), cholesteryl esters (CE), and total fatty acid composition. In situ fluorescent quantification of cytoplasmic membranes and LDs after cubosome treatment was also assessed in living cells loaded with Nile red, a fluorescent hydrophobic probe for the detection of polar and non-polar lipids (Greenspan et al., 1985).
Section snippets
Materials
Monoolein (RYLO MG 19 PHARMA, glycerol monooleate, 98.1 wt%; MO) was kindly provided by Danisco A/S, DK-7200, Grinsted, Denmark. Pluronic F108 (PEO132–PPO50–PEO132) (Fig. 1B), cholesterol, cholesteryl oleate (CO), cholesteryl arachidonate, standards of fatty acids, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (PC 16:0/16:0), 1,2-dioleoyl-sn-glycero-3-phosphocholine (PC 18:1/18:1), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Determination of MO level in cubosome formulation
Before any biological evaluation was performed, the amount of MO in cubosome formulation was monitored by HPLC determination. A MO level of 33.14 ± 2.56 μg/μL was determined in cubosome dispersion. Experiments in HeLa cells were performed using two concentrations of MO/PF108 cubosomes, 1:400 and 1:200, corresponding to 82.85 ± 6.41 and 165.69 ± 12.82 μg/mL of MO, respectively.
Cytotoxic activity (MTT assay)
MO-based cubosomes prepared with PF108 were tested for cytotoxicity (MTT assay) in HeLa cells. Fig. 2 shows the viability,
Discussion
NPs offer an extraordinary opportunity for application in pharmacology and medicine. However, NPs should no longer be viewed only as simple carriers for biomedical applications, since they can also play an active role in mediating biological effects (Kroll et al., 2009, Panariti et al., 2012, Przybytkowski et al., 2009). Actually, NPs interaction with living systems is currently originating growing interest under the perspective of improving drug delivery as well as implementing
Conclusions
This study showed that exposure of HeLa cells to MO-based cubosomes stabilized by Pluronic F108 (MO/PF108) can affect the intracellular metabolism of lipids. Results evidenced that MO/PF108 cubosome formulation was promptly taken up by the Hela cells and influenced the cellular lipid profile. Particularly, a significant synthesis of PL and TG rich in OA was observed along with a marked increase in the synthesis of intracellular membranes and accumulation of LDs. Given the importance of lipid
Funding sources
This work was supported by grants of the Regione Autonoma della Sardegna (CRP-59699).
Conflict of interest
The authors declare that they have no conflict of interest.
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