Comprehensive and comparative studies on nanocytotoxicity of glyceryl monooleate- and phytantriol-based lipid liquid crystalline nanoparticles

Abstract

Background: Lipid liquid crystalline nanoparticles (LLCNPs) emerge as a suitable system for drug and contrast agent delivery. In this regard due to their unique properties, they offer a solubility of a variety of active pharmaceutics with different polarities increasing their stability and the possibility of controlled delivery. Nevertheless, the most crucial aspect underlying the application of LLCNPs for drug or contrast agent delivery is the unequivocal assessment of their biocompatibility, including cytotoxicity, genotoxicity, and related aspects. Although studies regarding the cytotoxicity of LLCNPs prepared from various lipids and surfactants were conducted, the actual mechanism and its impact on the cells (both cancer and normal) are not entirely comprehended. Therefore, in this study, LLCNPs colloidal formulations were prepared from two most popular structure-forming lipids, i.e., glyceryl monooleate (GMO) and phytantriol (PHT) with different lipid content of 2 and 20 w/w%, and the surfactant Pluronic F-127 using the top-down approach for further comparison of their properties. Prepared formulations were subjected to physicochemical characterization and followed with in-depth biological characterization, which included cyto- and genotoxicity towards cervical cancer cells (HeLa) and human fibroblast cells (MSU 1.1), the evaluation of cytoskeleton integrity, intracellular reactive oxygen species (ROS) generation upon treatment with prepared LLCNPs and finally the identification of internalization pathways.

Results: Results denote the higher cytotoxicity of PHT-based nanoparticles on both cell lines on monolayers as well as cellular spheroids, what is in accordance with evaluation of ROS activity level and cytoskeleton integrity. Detected level of ROS in cells upon the treatment with LLCNPs indicates their insignificant contribution to the cellular redox balance for most concentrations, however distinct for GMO- and PHT-based LLCNPs. The disintegration of cytoskeleton after administration of LLCNPs implies the relation between LLCNPs and F-actin filaments. Additionally, the expression of four genes involved in DNA damage and important metabolic processes was analyzed, indicating concentration-dependent differences between PHT- and GMO-based LLCNPs.

Conclusions: Overall, GMO-based LLCNPs emerge as potentially more viable candidates for drug delivery systems as their impact on cells is not as deleterious as PHT-based as well as they were efficiently internalized by cell monolayers and 3D spheroids.

Keywords: Cellular internalization; Cubosomes; Cytoskeleton integrity; Cytotoxicity; Genotoxicity; Reactive oxygen species generation.

Fig. 1

Physicochemical characterization of fabricated LLCNPs. a Dynamic Light Scattering (DLS) intensity weighted particle size distribution curves of PHT and GMO LLCNPs, b Intensity weighted particle diameter of LLCNPs measured over a period of 60 days, c Scattering curves for PHT and GMO LLCNPs at 20 °C—the asterisk in SAXS plots indicates peak characteristic for liposomes. d Cryo-TEM images of I—GMO 2% and II—PHT 2% LLCNPs

Fig. 2

Viability studies with WST-1 assay. a HeLa and MSU 1.1 cells monolayers, b HeLa spheroids upon incubation with LLCNPs. The statistical analysis is included in Additional file 1: Table S1

Fig. 3

Visualization of reactive oxygen species generation in a HeLa and b MSU 1.1 cells upon treatment with LLCNPs. Scale bar: 20 µm

Fig. 4

Quantitative evaluation of reactive oxygen species (ROS) generation in HeLa and MSU 1.1 cells using DCFH-DA assay. Results presented as a relative DCF fluorescence intensity of ROS generation as a function of LLCNPs concentration, compared to HBSS and H₂O₂ controls, respectively negative and positive. Asterisks denotes the statistical significant difference compared with controls *—p ≤ 0.05, **—p ≤ 0.01

Fig. 5

Integrity of cytoskeleton in a HeLa and b MSU 1.1 cells upon treatment with LLCNPs. Scale bar: 20 µm

Fig. 6

Relative normalized expression of ACTB, CDK1, DHFR, GADD45A1 genes in HeLa and MSU 1.1 cells upon incubation with different LLCNPs formulations. Asterisks denotes the statistical significant difference compared with controls *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, ****—p ≤ 0.0001

Fig. 7

CLSM images of living HeLa and MSU 1.1 cells incubated with stained LLCNPs and with selected transport inhibitors. Red and green (lipid droplets) signals are LLCNPs, blue – nuclei. Scale bar: 10 μm

Fig. 8

Chemical structure of a glyceryl monooleate, b phytantriol and c Pluronic F127