A Novel Nanoconjugate of Landomycin A with C₆₀ Fullerene for Cancer Targeted Therapy: In Vitro Studies

Abstract

Introduction

Landomycins are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential. Literature data suggest that enhancement of the therapeutic activity of this drug may be achieved by means of creating specific drug delivery systems. Here we propose to adopt C₆₀ fullerene as flexible and stable nanocarrier for landomycin delivery into tumor cells.

Methods

The methods of molecular modelling, dynamic light scattering and Fourier transform infrared spectroscopy were used to study the assembly of C₆₀ fullerene and the anticancer drug Landomycin A (LA) in aqueous solution. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to human mesenchymal stem cells (hMSCs) using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) test and a live/dead assay. The morphology of the cells incubated with fullerene–drug nanoparticles and their uptake into target cells were studied by scanning electron microscopy and fluorescence light microscopy.

Results

The viability of primary cells (hMSCs, as a model for healthy cells) and cancer cell lines (human osteosarcoma cells, MG-63, and mouse mammary cells, 4T1, as models for cancer cells) was studied after incubation with water-soluble C₆₀ fullerenes, LA and the mixture C₆₀ + LA. The C₆₀ + LA nanocomplex in contrast to LA alone showed higher toxicity towards cancer cells and lower toxicity towards normal cells, whereas the water-soluble C₆₀ fullerenes at the same concentration were not toxic for the cells.

Conclusions

The obtained physico-chemical data indicate a complexation between the two compounds, leading to the formation of a C₆₀ + LA nanocomposite. It was concluded that immobilization of LA on C₆₀ fullerene enhances selectivity of action of this anticancer drug in vitro, indicating on possibility of further preclinical studies of novel C₆₀ + LA nanocomposites on animal tumor models.

Introduction

Landomycins (LAs) are a subgroup of angucycline antibiotics that are produced by Streptomyces bacteria and possess strong antineoplastic potential.12 All natural LAs share the same aglycon (landomycinone) and vary only in the number of sugar residues in their linear glycosidic chain containing only di- and trideoxysugars (β-d-olivose and α-l-rhodinose).4,12 It has been shown that LAs with longer saccharide chains have a higher activity towards tumor cell lines in vitro,19,45 and that LA with six sugar residues (LA-A) constitutes the most potent family member. Although the molecular mechanisms of the action of LAs are still poorly understood, recent studies on another family member, i.e., LA with three sugar residues (LA-E), have indicated the crucial role of specific extramitochondrial hydrogen peroxide production and early caspase-7 activation upstream of mitochondria.28

Enhancement of the therapeutic activity of anticancer drugs by means of specific drug delivery systems is considered as one of the most promising approaches in current pharmacology and medicine.15,26,47,48 Here we propose to adopt C₆₀ fullerene as flexible and stable nanocarrier for drug delivery into tumor cells.2,10,14,27,43 This idea is based on our previous extensive studies on the bioactivity of water-soluble pristine C₆₀ fullerenes which demonstrated a preferable targeting of tumor cells.20,32 This may be explained by their strong membranotropic properties8,29,44 as well as by a low toxicity towards normal cells and tissues of the organism.30,31,50 C₆₀ fullerene easily forms complexes with various compounds, including the well-known anticancer drugs doxorubicin (Dox) and cisplatin (Cis) as we demonstrated earlier.37^–39 Moreover, the conjugation of Dox and Cis to C₆₀ fullerene led to a significant increase of their cytotoxic activity in vitro and a therapeutic activity towards Lewis lung carcinoma in vivo,33^–35 thus confirming the role of C₆₀ fullerene as a versatile drug delivery platform that can be widely applied in clinical oncology.

Previously, by means of such powerful physico-chemical methods as the small-angle neutron scattering and atomic force microscopy, we accomplished a pilot study of intermolecular complexation and reported the effect of binding between C₆₀ fullerene and a novel anticancer drug candidate, i.e., LA (LA-A).40 In the present work this result was further investigated in more detail using the dynamic light scattering (DLS), Fourier transform infrared (FTIR) spectroscopy and molecular simulation techniques. The physico-chemical data were supplemented by in vitro studies of toxic effect of C₆₀ + LA nanocomplex towards normal and tumor cells and its penetration into target cells.

Experimental

Material Preparation

A stable pristine C₆₀ fullerene aqueous colloidal dispersion (C₆₀FAS) at the maximum concentration of 150 µg mL⁻¹ was prepared as described earlier,36,42 based on the original methodology which includes the transfer of C₆₀ molecules from toluene into an aqueous phase under ultrasonication.

In all experiments, we used LA powder (99.5% purity according to the high-performance liquid chromatography data) which was extracted from culture of Streptomyces globisporus S136 strain. LA was dissolved in methanol at the maximum concentration 200 µg mL⁻¹. Immobilization of LA on C₆₀ fullerene (i.e., the C₆₀ + LA mixture) was accomplished as follows40: the initial solution of C₆₀FAS and LA (with maximum concentration) were mixed in the volume ratio 1:1. The resulting mixture was ultrasonicated for 30 min, followed by magnetic stirring for 12 h by at room temperature.

DLS and ζ-Potential Determination

The size distribution and the ζ-Potential for C₆₀FAS and C₆₀ + LA mixture were determined by DLS on a Zetasizer Nano-ZS90 (Malvern, Worcestershire, UK) at room temperature. The instrument was equipped with a He–Ne laser (5 mW) operating at 633 nm. The results were analyzed under the Smoluchowski approximation.

FTIR Study

FTIR spectroscopy was used to study the structural organization of pristine C₆₀ fullerene with LA in aqueous solution. The FTIR spectra were recorded with a Perkin-Elmer BX-II spectrophotometer with a spectral resolution of 1 cm⁻¹ in the range of 400–4000 cm⁻¹. The samples were examined as thin film between two NaBr plates. For optimum signal-to-noise ratio, 64 scans were recorded per spectrum.

Structure Calculations

The procedure for structural analysis of the C₆₀ + LA nanocomplex corresponded to those used previously to study of the same nanocomplexes,40 except that in the present work we modelled the structure with maximum coverage of the C₆₀ fullerene surface by the ligand molecules. Briefly, the calculations of the spatial structure of C₆₀ + LA nanocomplex were performed by molecular mechanics using the X-PLOR software with the CHARMM27 force field. Quantum-mechanical calculations of partial atomic charges on C₆₀ fullerene and ligand atoms were performed with the Gaussian03 software within the framework of DFT (B3LYP) using a 6-31G* basis set by the Merz–Kollman method.

Cell Culture

The cytotoxic activity of the C₆₀ + LA nanocomplex was studied in vitro on human osteosarcoma cells (MG-63), mouse mammary cells (4T1), and primary human mesenchymal stem cells (hMSCs). The cell lines were obtained from the Collection of Microorganisms and Cell Cultures of the University of Duisburg-Essen. MG-63 and 4T1 cells were cultured in Dulbecco’s modified eagle medium (DMEM), supplemented with 10% fetal calf serum (FCS), 100 U mL⁻¹ penicillin, and 100 mg mL⁻¹ streptomycin at 37 °C in humidified atmosphere at 5% CO₂. hMSC were cultured in mesenchymal stem cell growth medium (MSCGM BulletKit™; Lonza). 12 h prior to uptake experiments, the cells were trypsinized and seeded in 48-well plates at a density of 25 × 10³ cells per well in 0.25 mL DMEM with FCS for MG-63 and 4T1 cells and with MSCGM for hMSCs.

Scanning Electron Microscopy (SEM)

The attachment of free and LA-immobilized C₆₀ fullerene to the membrane of MG-63 cells, 4T1 cells, and hMSCs was visualized by SEM. Cells were cultured for 1 h either with or without C₆₀FAS, and rinsed twice with phosphate-buffered saline (PBS). Then the cells were fixed with 3.7% glutaraldehyde (Sigma-Aldrich, Taufkirchen, Germany) in PBS for 15 min. After a 2-fold washing with PBS, the fixed cells were dehydrated with an ascending sequence of ethanol (20, 40, 60, 80 and 96–98%). Subsequently, after evaporation of ethanol, the samples were left for 2 h at 37 °C and then analyzed by SEM. SEM was performed with an ESEM FEI Quanta 400 instrument with gold/palladium-sputtered samples.

Fluorescence Microscopy

The uptake studies of C₆₀FAS (75 μg mL⁻¹), of LA (100 μg mL⁻¹), and of the C₆₀ + LA mixture (75 + 100 μg mL⁻¹) were carried out as follows. The cells were incubated with 5 μL dispersion/solution of the indicated compounds for 3 and 24 h, respectively. Immediately after the indicated time, the cells were washed three times with PBS to remove all dissolved compounds that were not attached to the cells. The cellular uptake was measured by transmission light microscopy and fluorescence microscopy with a Keyence Biorevo BZ-9000 instrument (Osaka, Japan), equipped with filters for 4′, 6 diamidino 2 phenylindole (DAPI, EX 360/40, DM 400, BA 460/50) at ×20 magnification. C₆₀ + LA nanocomplexes were visible as blue fluorescing dots.

MTT and Live/Dead Assay

The cell viability was analyzed by an 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay 72 h after the incubation of MG-63 cells, 4T1 cells and hMSCs with C₆₀FAS (75 μg mL⁻¹), LA (100 μg mL⁻¹) or the mixture of C₆₀ + LA (75 + 100 μg mL⁻¹). The following volumes of dispersion were added to the cells (total volume of 250 µL per well): 0.5, 2.5, 5 or 10 μL, which corresponds to the following concentrations: 0.15, 0.75, 1.5, 3.0 μg mL⁻¹ of C₆₀FAS, 0.2, 1.0, 2.0 and 4.0 μg mL⁻¹ of LA, 0.15 + 0.2, 0.75 + 1.0, 1.5 + 2.0 and 3.0 + 4.0 μg mL⁻¹ of C₆₀ + LA mixture. The used ratio of concentrations in the studied mixtures, presumably, corresponds to the optimum binding efficiency of LA to C₆₀ fullerene clusters as evidenced by structural analysis (see below).

MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Sigma, Taufkirchen, Germany) was dissolved in PBS (5 mg mL⁻¹) and then diluted to 1 mg mL⁻¹ in the cell culture medium. The culture medium of the incubated cells was replaced by 300 µL of the MTT solution. The cells were incubated for 1 h at 37 °C under 5% CO₂ in humidified atmosphere. 300 µL of dimethyl sulfoxide (DMSO) was added to the cells. After 30 min, a 100 µL aliquot was taken for spectrophotometric analysis with a Multiscan FC instrument (Thermo Fisher Scientific, Vantaa, Finland) at λ = 570 nm. The absorption of incubated cells was normalized to that of control (untreated) cells, thereby indicating the relative level of cell viability. Each concentration of the studied compounds was run in triplicate and normalized to blank controls, containing the equivalent volume of culture medium.

A live/dead assay was carried out according to the following protocol. 72 h after the incubation of MG-63 cells, 4T1 cells, and hMSCs with 5 μL of C₆₀FAS (75 μg mL⁻¹), LA (100 μg mL⁻¹) and C₆₀ + LA mixture (75 + 100 μg mL⁻¹), respectively, the cells were washed with PBS and stained with a live/dead viability/cytotoxicity assay for mammalian cells (L3224, Invitrogen Co.) to evaluate the cell viability. 150 μL of the calcein AM and ethidium homodimer-1 working solution were directly added to the cells. Afterwards, the cells were subsequently incubated for 30 min at 37 °C. After incubation, the stained cells were imaged by fluorescence microscopy (Keyence). The live/dead kit determines the cell viability based on the cell membrane integrity. Living cells are stained by calcein AM which emits a green fluorescence (517 nm) when excited by blue light (494 nm), whereas dead cells are stained by EthD-1 which emits a red fluorescence (617 nm) when excited by green light (528 nm). All experiments were carried out in triplicate.

Statistics

Statistical analysis was performed by conventional methods of variation statistics. The significance of the differences between the control and experimental measurements was estimated by Student’s t test using the Origin 8.0 software (OriginLab Corporation, USA). The difference between the compared values was considered to be significant at p < 0.05.

Results and Discussion

Material Characterization

It has been established that the size of C₆₀ fullerene particles directly correlates with their biodistribution and toxicity.21,30,31,50 Depending on the size, C₆₀ fullerene particles can penetrate through the plasma membrane into the cell or be adsorbed on the surface of the membrane.8,9,29,44 Therefore, DLS measurements of C₆₀FAS and C₆₀ + LA dispersions were performed (Table 1).

Table 1.

The hydrodynamic particle diameter, the PDI value, and the ζ-Potential for C₆₀FAS and C₆₀ + LA aggregates at room temperature in aqueous dispersion, measured by DLS method.

Samples	Diameter (nm)	PDI	ζ-Potential (mV)
C60FAS	184 ± 18	0.209	− 17.0 ± 2.0
C60 + LA mixture	190 ± 20	0.219	− 1.0 ± 0.1

C₆₀ fullerenes and C₆₀ fullerenes with LA form particles (aggregates) in water with an average hydrodynamic size 184 and 190 nm, respectively, in good agreement with previous results.40 The obtained result means that presence of LA solution only slightly affects the distribution of C₆₀ fullerene aggregates in aqueous solution. The value of polydispersity index (PDI, about 0.21) indicates a low polydispersity of these systems. The magnitude of ζ-Potential measured for C₆₀ + LA mixture shifted to -1 mV from the initial value of − 17 mV measured for C₆₀FAS, indicating an interaction between C₆₀ fullerene and LA.

The calculated structure of the C₆₀ + LA nanocomplex (Fig. 1) indicates the possibility of binding of up to two LA molecules to the surface of one C₆₀ fullerene molecule, suggesting a favorable hydrophobic complexation effect. Both LA ligands are stabilized by π-stacking with the C₆₀ molecule surface which is known as the dominant force in C₆₀ fullerene–aromatic interactions.33^–35,37^–40 Importantly, the maximal binding ratio 1:2 provides rough estimate of the ratio of concentrations (1:1…1:2) to be further used in in vitro experiments and corresponding to optimal binding efficacy of LA to fullerene clusters in solution.

Figure 1.

Calculated structure of 1:2 C₆₀ + LA nanocomplex.

Further characterization of the C₆₀ + LA interaction was carried out by FTIR spectroscopy. The FTIR spectrum of C₆₀ fullerene with LA mixture is shown in Fig. 2 together with the reference spectra of C₆₀FAS and LA. The spectrum of C₆₀FAS exhibits four intense peaks at 1423, 1180, 576, and 526 cm⁻¹ attributed to the C–C vibrational modes of the C₆₀ molecule and weak bands at 3372 and 1666 cm⁻¹, corresponding to water hydrogen-bonded O–H stretching and bending, respectively; the broad intense band near 1107 cm⁻¹ is attributed to C–OH stretching; a weak band at 2957–2850 cm⁻¹, corresponds to C–H stretching.18 Two strong broad bands centered at 3410 and 3373 cm⁻¹ corresponding to hydrogen-bonded O–H stretching [ν(OH)] of the methanol and a weak band at 1734 cm⁻¹ [δ(OH)] were observed in the FTIR spectra of the LA and C₆₀ + LA mixture, respectively, because the measurement was performed on alcohol substance. The O–H stretching and CH₃ stretch bands of the LA in the FTIR spectra of the methanol solution and C₆₀ + LA mixture had low intensities and overlapped with bands of the solvent. Aromatic C=C stretches of LA at 1368 and 1450 cm⁻¹ had low intensities and overlapped with bands of the C–C vibrational modes and C–O–H bending of the C₆₀FAS system.18 The group of peaks from 1111 to 975 cm⁻¹ centered at 1055 cm⁻¹, corresponding to dialkyl- and alkyl–aryl ether C–O stretches of LA. Also, the characteristic strong band at 1610 cm⁻¹ assigned to a C=O stretch conjugated with the aromatic system is found both for the LA and C₆₀ + LA.

Figure 2.

FTIR spectra of C₆₀FAS, LA and C₆₀ + LA mixture.

It is known that the stacking of aromatic surfaces results in a high-frequency shift of C=O functional groups by few cm⁻¹.7 Such shifts were initially expected for the C₆₀ + LA interaction, which, however, they were not observable on the measured FTIR spectrum of the mixture due to strong line broadening (Fig. 2). However, the presence of C–OH vibrations in FTIR spectrum of C₆₀FAS provides another important source of C₆₀ + LA nanocomplex stabilization due to intermolecular hydrogen bonding to proton acceptor groups of the ligand. Note, such additional stabilization is well known for aromatic–aromatic π-stacked complexes in solution.6

In Vitro Experiments

Pure LA and the C₆₀ + LA mixture showed a clear concentration-dependent cytotoxic effect towards MG-63 cells, 4T1 cells, and hMSCs after 72 h incubation (Fig. 3). No toxic effects of C₆₀FAS towards these cell lines were observed even at the highest concentration. The same result was observed for C₆₀FAS in vitro against to normal cells (thymocytes) and cancer cells (Ehrlich ascitic carcinoma, leukemia L1210 and HeLa).30,31,50 LA alone was toxic for all three cell types, and its cytotoxicity increased with increasing LA concentration. In particular, IC₅₀ values for LA were 1.81, 0.55 and 0.88 µg mL⁻¹ for MG-63, 4T1 cells and hMSCs, respectively. Interestingly, at the increasing concentration of the C₆₀ + LA mixture, we observed a higher toxicity for cancer cells and a weakly pronounced toxicity (up to 40%) for hMSCs. These data were also confirmed by analysis of IC₅₀ value of C₆₀ + LA nanocomplex: it decreased 2.5-fold for MG-63 cells (from 1.81 down to 0.74 µg mL⁻¹), but gradually increased 2-fold for hMSCs (from 0.88 up to 1.58 µg mL⁻¹). The live/dead assay gave the same results: C₆₀FAS was not toxic for the cells, LA killed almost all cells and the C₆₀ + LA mixture, in contrast to LA alone, showed high toxicity for cancer cells and low toxicity for hMSCs (Fig. 4).

Figure 3.

MTT assay for MG-63 cells (A), 4T1 cells (B) and hMSCs (C) after 72 h of incubation with C₆₀FAS, LA and C₆₀ + LA mixture at different concentrations: 0.15, 0.2 and 0.15 + 0.2 μg mL⁻¹ (a), 0.75, 1.0 and 0.75 + 1.0 μg mL⁻¹ (b), 1.5, 2.0 and 1.5 + 2.0 μg mL⁻¹ (c), 3.0, 4.0 and 3.0 + 4.0 μg mL⁻¹ (d). Data are given relative to the untreated control samples (p < 0.05) and represent the mean ± SD of three independent experiments.

Figure 4.

Representative live/dead staining of MG-63 cells, 4T1 cells and hMSCs after 72 h of incubation with C₆₀FAS (1.5 μg mL⁻¹), LA (2.0 μg mL⁻¹) or C₆₀ + LA mixture (1.5 + 2.0 μg mL⁻¹) and mock (untreated cells). Scale bar 20 µm.

The SEM micrographs of MG-63 cells, 4T1 cells, and hMSCs, incubated with C₆₀ fullerene, LA and C₆₀ + LA mixture, as well as without any additions (as a control) are shown in Fig. 5. The SEM images of single cells were chosen to represent the whole population in each group as all cells in the group had the same morphology. The SEM images underline the results obtained from live/dead assay, i.e., at a LA concentration of 2 μg mL⁻¹ both types of cancer cells as well as the primary cells were dead in contrast to the C₆₀ + LA mixture, where we observed a high toxic effect for cancer cells, but only a low toxicity for primary cells.

Figure 5.

SEM micrographs of MG-63 cells, 4T1 cells and hMSCs after 72 h of incubation with C₆₀FAS (1.5 μg mL⁻¹), LA (2.0 μg mL⁻¹) or C₆₀ + LA mixture (1.5 + 2.0 μg mL⁻¹) and mock (untreated cells).

The membranotropic effect of LA and C₆₀ + LA nanoparticles on MG-63 cells and hMSCs (as a representative model for cancer and primary cells) was studied by fluorescence microscopy. In the Fig. 6, the uptake of LA and C₆₀ + LA nanocomplex into cells after 2 h of incubation is shown. It was revealed that LA weakly fluoresces at DAPI channel (blue) of fluorescence microscope in the hMSC and MG-63 cells. In the control (untreated cells) no blue fluorescence was detected (data not shown). Immobilization of LA on C₆₀ fullerene leads to enhancement of LA fluorescence inside the cells in a concentration-dependent manner. Thus, C₆₀ fullerene enhances LA’s entry inside tumor cells due to its membranotropic activity, thus improving cytotoxic activity of these nanocomplexes. Taking into consideration that similar effects of C₆₀ fullerene were already observed by us for C₆₀ + Dox and C₆₀ + Cis nanocomplexes, one may conclude that enhancement of drug uptake into tumor cells is the unique feature of C₆₀ fullerene, which may be of high importance for further pre-clinical trials of the C₆₀ fullerene–drug nanocomplexes.25,41

Figure 6.

Transmission light microscopy and fluorescence microscopy of MG-63 and hMSC cells after 2 h of incubation with LA (2.0 μg mL⁻¹) and C₆₀ + LA mixture (1.5 + 2.0 μg mL⁻¹). The blue fluorescence indicates the co-localization of LA and C₆₀ + LA nanocomplex on the cells. Note that C₆₀ fullerene alone is not fluorescent. Scale bar 20 µm.

The peculiarity of the biological effect of the water-soluble C₆₀ fullerenes is their ability to selectively damage of the tumor cells, whereas they do not affect the normal cells.23,46 On the other hand, C₆₀ fullerene, as a carrier of antitumor drug, thanks to its powerful antioxidant properties,5,51 effectively reduces the side (toxic) effects of drug relative to the normal cells.1,35

It is known that the blood vessels in most solid tumors are characterized by intense angiogenesis, high density and increased vascular permeability, defective vascular networks and defective or depressed lymphatic drainage in the intratissue space of the tumor,22 which allows nanocompounds to penetrate, accumulate and hold in tumors. In addition, due to impaired endocytosis, the tumor cells are able to accumulate more and maintain longer, than the normal cells, exogenous compounds, especially with high molecular weight.49 Therefore, it can be assumed that with an increase in the size/mass of C₆₀ molecule due to its complexation with the chemotherapeutic drug and simultaneous association with the serum proteins, there is a passive accumulation of these nanoparticles in the tumor through the enhanced permeability and retention (EPR) effect in the tumor vasculature.3,11,13,24 In this regard, the recent study16 clearly demonstrated the feasibility of tracking and quantifying the delivery kinetics and intratumoral biodistribution of C₆₀ fullerene-based drug delivery platform, consistent with the EPR effect on short timescales and passive transport to tumors. On the other hand, it was found that C₆₀ fullerenes increase the sensitivity of tumor cells to the action of cytostatics due to their interaction and non-recognition by ABC-transporters.17,34

Conclusion

A complexation between pristine C₆₀ fullerene and LA molecule in aqueous solution was characterized by molecular modelling, DLS and FTIR techniques. Cytotoxic activity of this nanocomplex was studied in vitro towards two cancer cell lines in comparison to hMSCs using MTT test and a live/dead assay. It was shown that C₆₀ fullerene nanoparticles were non-toxic for the cells, LA at the concentration of 2 μg mL⁻¹ killed all tested cell lines, while its nanocomplex with C₆₀ fullerene at the same concentration of LA demonstrated high toxicity for cancer cells and much lower toxicity for mesenchymal stem cells. The same results were obtained by live/dead assay: C₆₀ + LA mixture had shown high toxicity for cancer cells, but not for the mesenchymal stem cells. These data were supported by fluorescence microscopy studies, which have shown higher uptake of C₆₀ + LA nanocomplex by tumor cells, and lower one by mesenchymal stem cells. The obtained results indicate on importance of using C₆₀ fullerene as a nanoplatform for drug delivery, and, in particular, huge potential of C₆₀ + LA nanocomplex as a novel anticancer agent.

Abbreviations

4T1

Mouse mammary cells

C₆₀FAS

C₆₀ fullerene aqueous solution

Cis

Cisplatin

DAPI

4′, 6 Diamidino 2 phenylindole

DLS

Dynamic light scattering

DMEM

Dulbecco’s modified eagle medium

DMSO

Dimethyl sulfoxide

Dox

Doxorubicin

EPR

Enhanced permeability and retention

FCS

Fetal calf serum

FTIR

Fourier transform infrared spectroscopy

hMSCs

Human mesenchymal stem cells

LA

Landomycin A

MG-63

Human osteosarcoma cells

MTT

3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide

PBS

Phosphate-buffered saline

PDI

Polydispersity index

SEM

Scanning electron microscopy

Authors’ Contributions

The work presented here was carried out in collaboration between all the authors. RP, JR, VO and YP created and characterized nanomaterials. VB and VS performed in vitro and fluorescence microscopy studies. OL and SP characterized nanomaterials using FTIR analysis. ME performed the computer simulations. UR synthesized and characterized C₆₀FAS. M. Epple and YP coordinated the experimental work, analyzed the data, performed the statistical analysis, and wrote the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.