Elsevier

Chemistry and Physics of Lipids

Volume 191, October 2015, Pages 96-105
Chemistry and Physics of Lipids

Monoolein-based cubosomes affect lipid profile in HeLa cells

https://doi.org/10.1016/j.chemphyslip.2015.08.017Get rights and content

Highlights

Abstract

Monoolein-based cubosomes are promising drug delivery nanocarriers for theranostic purposes. Nevertheless, a small amount of research has been undertaken to investigate the impact of these biocompatible nanoparticles on cell lipid profile. The purpose of the present investigation was to explore changes in lipid components occurring in human carcinoma HeLa cells when exposed to short-term treatments (2 and 4 h) with monoolein-based cubosomes stabilized by Pluronic F108 (MO/PF108). A combination of TLC and reversed-phase HPLC with DAD and ELSD detection was performed to analyze cell total fatty acid profile and levels of phospholipids, free cholesterol, triacylglycerols, and cholesteryl esters. The treatments with MO/PF108 cubosomes, at non-cytotoxic concentration (83 μg/mL of MO), affected HeLa fatty acid profile, and a significant increase in the level of oleic acid 18:1 n-9 was observed in treated cells after lipid component saponification. Nanoparticle uptake modulated HeLa cell lipid composition, inducing a remarkable incorporation of oleic acid in the phospholipid and triacylglycerol fractions, whereas no changes were observed in the cellular levels of free cholesterol and cholesteryl oleate. Moreover, cell-based fluorescent measurements of intracellular membranes and lipid droplet content were assessed on cubosome-treated cells with an alternative technique using Nile red staining. A significant increase in the amount of the intracellular membranes and mostly in the cytoplasmic lipid droplets was detected, confirming that monoolein-based cubosome treatment influences the synthesis of intracellular membranes and accumulation of lipid droplets.

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.

References (41)

  • M.A. Maier et al.

    Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics

    Mol. Ther.

    (2013)
  • X. Mulet et al.

    Advances in drug delivery and medical imaging using colloidal lyotropic liquid crystalline dispersions

    J. Colloid Interface Sci.

    (2013)
  • S. Murgia et al.

    Addition of hydrophilic and lipophilic compounds of biological relevance to the monoolein/water system II—13C NMR relaxation study

    Chem. Phys. Lipids

    (2001)
  • S. Murgia et al.

    Cubosome formulations stabilized by a dansyl-conjugated block copolymer for possible nanomedicine applications

    Colloids Surf. B

    (2015)
  • A. Rosa et al.

    Potential anti-tumor effects of Mugil cephalus processed roe extracts on colon cancer cells

    Food Chem. Toxicol.

    (2013)
  • C.J. Schiller et al.

    Assessment of viability of hepatocytes in suspension using the MTT assay

    Toxicol. In Vitro

    (1992)
  • J.E. Vance

    MAM (mitochondria-associated membranes) in mammalian cells: lipids and beyond

    Biochim. Biophys. Acta

    (2014)
  • C. Caltagirone et al.

    Cancer-cell-targeted theranostic cubosomes

    Langmuir

    (2014)
  • A.M. Falchi et al.

    Effects of monoolein-based cubosome formulations on lipid droplets and mitochondria of HeLa cells

    Toxicol. Res.

    (2015)
  • A. Galli et al.

    High-level expression of rat class I alcohol dehydrogenase is sufficient for ethanol-induced fat accumulation in transduced HeLa cells

    Hepatology

    (1999)
  • Cited by (0)

    View full text