The application of nanomaterials in the fields of medicine and biotechnology is of enormous interest, particularly in the areas where traditional solutions have failed. Unfortunately, there is very little information on how to optimize the preparation of nanomaterials for their use in cell culture and on the effects that these can trigger on standard cellular systems. These data are pivotal in nanobiotechnology for the development of different applications and to evaluate/compare the cytotoxicity among the different nanomaterials or studies. The lack of information drives many laboratories to waste resources performing redundant comparative tests that often lead to partial answers due to differences in (i) the nature of the start-up material, (ii) the preparation, (iii) functionalization, (iv) resuspension, (v) the stability/dose of the nanomaterial, etc. These variations in addition to the different analytical systems contribute to the artefactual interpretation of the effects of nanomaterials and to inconsistent conclusions between different laboratories. Here, we present a brief review of a wide range of nanomaterials (nanotubes, various nanoparticles, graphene oxide, and liposomes) with HeLa cells as a reference cellular system. These human cells, widely used as cellular models for many studies, represent a reference system for comparative studies between different nanomaterials or conditions and, in the last term, between different laboratories.
Nanomaterials offer revolutionary solutions to traditional problems, and thus, they have been incorporated into many different consumer goods including many for human consumption such as cosmetics, biotechnological and pharmacological products, medicines, or food additives. Unfortunately, major developments are never exempt of associated problems. Nanomaterials have been connected with all types of toxicological, cumulative, or environmental problems [
Cellular models are very convenient because they do not require complex laboratory facilities and can provide pivotal information on the toxicological effects of nanomaterials; furthermore, nowadays, there are many cellular models to investigate these interactions with nanomaterials. In fact, many studies have been carried out using cells of different origins and diverse natures. However, this poorly protocolised research has resulted in the production of confusing and incoherent data that result in chaos when it comes to understanding and comparing the effect of a particular nanomaterial at the same dose in a unique cellular system.
In our laboratories, we have been studying the
There are many examples in the literature where cells exposed to the same nanomaterial respond differently. This is the case of carbon nanotubes where the reported effects range from innocuity to very acute toxicological or long-term accumulative effects [
For all these reasons, it seems necessary to carry out standard tests where a single cell type is exposed to the same concentration of different nanomaterials, functionalized, and processed identically, following the same protocol. This test allows the direct comparison of the effect of the different nanomaterials on a unique system
On the other hand, this cell line has been used in many laboratories on the assumption that it comprises putatively homogeneous clonal cell population. However, recent studies demonstrate that HeLa cells are heterogeneous [
Different nanomaterials were used in this work. High-purity MWCNTs were obtained from Nanocyl NC3100™. These nanotubes have been fully characterized in previous publications [
Nanomaterials were resuspended and functionalized in a saline solution containing 30% fetal bovine serum (FBS, Gibco) by mild probe sonication (3-5 cycles, 2-5
HeLa cells (from the European Molecular Biology Laboratory Cell Bank, passage 10) were cultured under standard conditions in Minimum Essential Medium (MEM) containing 10% FBS and antibiotics (Gibco, Thermo Fisher Scientific). Phase contrast micrographs were taken at different time points using a Progress CT5 (Jenoptik) digital camera coupled to a Nikon Eclipse TS100-F. Cell viability assessments were performed using a standard trypan blue assay. The cell cycle distribution was analysed by flow cytometry using a Muse® Cell Analyzer (Merck KGaA) following the manufacturer’s instructions. Immunostaining was performed on cells fixed in 4% paraformaldehyde. Phalloidin-tetramethylrhodamine B isothiocyanate, Hoechst dye (bisbenzimide), and Acridine Orange hemi (zinc chloride) salt (all from Sigma-Aldrich) were used to stain actin, DNA, and cytoplasm, respectively. Microtubules were immunostained with the B512 anti-
Carbon nanotubes (CNTs) represent a class of highly versatile materials that display very interesting mechanical, thermal, electronic, and biological properties [
Multiwalled carbon nanotube (MWCNT) interaction with HeLa cells. (a) Diagram of their internalization and endo-lysosomal escape. (b) Low- and high-resolution TEM images of MWCNTs. (c) Phase contrast images of the control and 72 h MWCNT-exposed HeLa cells. (d) Asymmetric triple mitosis representative of the biomechanical defects triggered by MWCNTs in the microtubule cytoskeletal machinery. (e) MWCNTs treatment resulted in a drop in the number of cells at the G1 stage (nondividing cells, represented in light blue) and a rise of cells at S (DNA synthesis), G2 stage (mitotic), polyploidy (P, aberrant genomic load), and apoptosis. Changes in the cell cycle after 72 h incubation with MWCNTs are indicative of cell cycle blockage.
Silica particles are traditionally considered to be quite biocompatible and have been used as a therapy delivery system due to their interesting physico-chemical properties [
Silica particle interaction with HeLa cells. (a) Schematic diagram of silica particles processing in HeLa cells. Particles are internalized in cells via endocytosis. In endo-lysosomes, their biocorona is degraded, and stripped particles are finally exocytosed. ((b), A) TEM characterization of the as-prepared silica particles. ((b), B) Confocal image demonstrating silica particles inside HeLa cells. (c) Confocal image where the exocytosed silica particles are detected (red arrows). The accompanying histogram shows the percentage of extracellular particles after 15
Carbon nanotubes can be used to coat silica particles to trigger the lysosomal exit imitating viral escape mechanisms [
CNT-silica particle interaction with HeLa cells. (a) Schematic diagram of CNT-silica particle trajectory in HeLa cells. Once these particles are internalized via endocytosis, the CNT biocorona is degraded. Stripped nanotubes interact with the lysosomal membrane and escape the endo-lysosomal compartment. (b) TEM images of some representative CNT-silica particles. ((c), A) Confocal microscopy images demonstrating internalized particles in HeLa cells (red arrows). ((c), B) TEM micrograph of a section of a HeLa cell cytoplasm where the CNTs of the coating are observed piercing the endo-lysosomal membrane. ((d), A) Percentage of live cells after 24 h, 48 h, and 72 h of exposure to 50
Graphene oxide is a nanomaterial that, up to date, has been reported to be quite biocompatible in different systems
GO interaction with HeLa cells. (a) Schematic diagram of functionalized GO contacting with cellular surface receptors and invading the cell via endocytosis. Internalized GO flakes are progressively degraded in lysosomes. (b) TEM characterization of GO flakes. (c) Confocal microscopy image of HeLa cells exposed to 50
Liposomes are widely used as delivery systems to transfer drugs, proteins, or nucleic acids into target cells. These nanovesicles made up of a lipid bilayer can encapsulate different types of therapies into an inner aqueous phase or lipid bilayer and are considered, in general, very biocompatible nanostructures [
Cationic liposome interaction with HeLa cells. (a) Graphic illustration of the intracellular cycle of cationic liposomes in HeLa cells. (b) Confocal microscopy image of HeLa cells exposed to DiI-labelled cationic liposomes (red channel, in boxes). (c) TEM image of the as-prepared cationic liposomes. (d) HeLa cell viability after cationic liposome exposure. (e) Phase contrast image of HeLa cells exposed to the cationic liposomes for 72 h. (f) Comparative study of the cell cycle between HeLa cells with CLP by flow cytometry.
Titanium dioxide (TiO2) nanoparticles are some of the most commonly manufactured nanomaterials that are extensively used as components of paints, cosmetics, food, and many other consumer products [
Titanium oxide nanoparticle interaction with HeLa cells. (a) Graphic illustration of the intracellular trajectory of TiO2 nanoparticles in HeLa cells. (b) Confocal microscopy images of HeLa cells exposed to TiO2 nanoparticles where no morphological changes are observed. (c) TEM images of the pristine TiO2 nanoparticles. (d) Cell survival after exposure to 50
HeLa cells that are subjected to treatment with ZnO display a very acute phenotype of cytotoxicity due to the dissolution of the ZnO nanoparticles inside the lysosomes [
Interaction of ZnO nanoparticles and nanowires with HeLa cells. (a) Graphic illustration of the intracellular cycle of ZnO nanomaterials in HeLa cells. (b) TEM characterization of ZnO nanoparticles (A) and nanowires (B). (c) Phase contrast images of HeLa cells exposed to 50
ZnO:Co2+ nanowires caused very similar effect to ZnO nanoparticles despite the different morphology and composition. These nanomaterials functionalized with serum proteins interact with membrane receptors, trigger endocytosis, and finally, dissolve in the lysosomes virtually identical to ZnO nanoparticles [
Our work shows how the toxicological effects of nanomaterials can result from the morphology of the nanomaterials and/or their composition. In this review, we report several cases that illustrate these behaviours. In the case of carbon nanotubes and GO, it is the morphology rather than the composition factor that triggers the cytotoxic response in the HeLa cells. However, this is not a universal dogma, for ZnO-based nanomaterials, morphology is less important than chemistry, and it is its composition and their chemical properties which trigger the cytotoxic effect in that case. Also, this work demonstrates how nanomaterials can produce unpredictable consequences in human HeLa cells; even if we can know the composition and morphology of nanomaterials, a complete cytotoxic study should be performed in each case.
Our results show that although all employed nanomaterials interact with cells by receptor-mediated endocytosis, the route that they follow once inside the cell is not the same. As we can see, our study reinforces the idea that it is necessary to develop specific tests for each nanomaterial, since it is not possible to anticipate the cytotoxic effects and/or the interaction with cells and tissues.
This exhaustive study constitutes an extraordinary tool for the modelling of more complex structures, incorporating materials endowed with magnetic, optical, or catalytic functionalities. In fact, the carbon nanotubes provide a high surface, which makes easier the adsorption of many different ligands, together with other porous or hollow materials like mesoporous SiO2 or liposomes. Also, we can take advantage of other materials that are innocuous to these cells like TiO2 in order to improve the biostability. This can increase the applications of these nanomaterials as drug delivery systems, therapy or diagnostic.
Nanomedicine and in particular the study of these interactions between nanomaterials and biological systems is a field in constant development and evolution that changes every day making the designs and the possibilities almost endless.
All data used to support the findings of this study are included within the article and can be provided by the corresponding author upon request.
The authors declare that there is no conflict of interest regarding the publication of this paper.
CRL is funded by FJCI-2015-25306; LGL is funded by CD17/00105; LVF is funded by FPU 15/06881.
This work has been supported by the Spanish MINECO and European FEDER under Project ref. PI16/00496, the NanoBioApp Network Ref. MINECO-17-MAT2016-81955-REDT. We thank IDIVAL for INNVAL15/16, INNVAL 17/11, PREVAL 16/03, 16/02, and the Raman4clinics BMBS COST Actions BM1401 and TD1402. We also thank Débora Muñoz for her technical assistance. We are grateful to the Nikon A1R Laser Microscopy Unit and the TEM Unit of the IDIVAL Institute.