In-vitro cytotoxicity assessment of carbon-nanodot-conjugated Fe-aminoclay (CD-FeAC) and its bio-imaging applications

We have investigated the cytotoxic assay of Fe-aminoclay (FeAC) nanoparticles (NPs) and simultaneous imaging in HeLa cells by photoluminescent carbon nanodots (CD) conjugation. Non-cytotoxic, photostable, and CD NPs are conjugated with cationic FeAC NPs where CD NPs play a role in bio-imaging and FeAC NPs act as a substrate for CD conjugation and help to uptake of NPs into cancer cells due to positively charged surface of FeAC NPs in physiological media. As increase of CD-FeAC NPs loading in HeLa cell in vitro, it showed slight cytotoxicity at 1000 μg/mL but no cytotoxicity for normal cells up to concentration of 1000 μg/mL confirmed by two 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays, with further observations by 4′,6-diamidino-2-phenylindole (DAPI) stained confocal microscopy images, possessing that CD-FeAC NPs can be used as potential drug delivery platforms in cancer cells with simultaneous imaging.Graphical abstract CD conjugation with organo-building blocks of delaminated FeAC NPs Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0151-z) contains supplementary material, which is available to authorized users.

Recently, organo-building blocks of Mg-and Ca-aminoclays were tested for possible use as drug-delivery carriers, and were found to result in neither cytotoxicity nor inflammation [29]. Further, protonated clusters of Mg-aminoclay with positively charged zeta potential in the wide pH range of 2.0-12.0 [30] were tested as biodistribution and elimination pathways in in vivo mice after Cy 5.0 conjugation with organo-building blocks in delaminated Mg-aminoclay. The results showed fast elimination or excretion of Mg-aminoclay in mice after oral or intravenous injection, respectively, without toxicity [31]. With the exceptions of transparent Mg-and Ca-aminoclays in aqueous solutions, other colored aminoclays have not been tested for cytotoxicity to determine the feasibility of their use in biomedical applications. Importantly, the lack or weak fluorescent-emission intensity of aminoclays has driven research to explore fluorescent imaging for promising drug-delivery-carrier and simultaneous bio-imaging applications in diagnostics and therapeutics.

Rudimental characteristics of FeAC, CD, and CD-FeAC NPs
The PL spectra of FeAC, CD, and CD-FeAC NPs at 290-410 nm excitation wavelengths were recorded with emission spectra (Fig. 2). The PL intensity of FeAC NPs was very weak (Fig. 2a), whereas CD NPs, at an 8.4 % internal quantum yield, showed suitable PL (Fig. 2b). The CD-FeAC NPs also showed good PL data for the purposes of bio-imaging (Fig. 2c). In corresponding Raman spectra, due to the stong PL interference in the CD NPs, CD-FeAC NPs peaks showed indistinguishable D and G bands (Additional file 1: Figure S1) [43]. Transmission electron microscopy (TEM) images of FeAC, CD, and CD-FeAC NPs dispersed in aqueous solution showed successful conjugation of CD NPs with FeAC NPs, with clear contrasts (Fig. 3), compared to only carbon coated copper grid. FeAC NPs displayed the amorphous phase in the entire several-layer morphology, with distinct contrasts ( Fig. 3a, b), which result is consistent with the relevant previous study [40][41][42] and CD NPs manifested 2-5 nm spherical and semicrystalline sizes but with some aggregated NPs with ~20 nm size (Fig. 3c, d), in the obtained atomic force microscopy (AFM) image showing a rough root mean square (RMS) of 1.865 (Additional file 1: Figure S2). As for CD-FeAC NPs, CD NPs seemed to be uniformly distributed in the FeAC sheets, and FeAC NPs had an dispersion ability of CD NPs (Fig. 3e, f ), especially in light of the containment of CD NPs in the FeAC matrix [44].
For additional information relevant to in vivo mice experimentation and further clinical trials, the aqueous behaviours of FeAC, CD, and CD-FeAC NPs also are needed. Thus, their surface chemistries (e.g., zeta potentials) and hydrodynamic sizes were measured in PBS buffer and serum-free RPMI media at neutral pH ( Table 2). The surface charges of FeAC NPs in the PBS buffer and serum-free RPMI media were, respectively, approximately +8 and +2.3 mV of the zeta potentials, whereas that of CD was approximately -30 mV. These results were ascribed, respectively, to the abundance of protonated amine groups (i.e., cationic clusters) and carboxylated groups. Accordingly, the zeta potentials of CD-FeAC NPs in PBS buffer and nutrient media were approximately −4.0 and −7.0 mV, respectivley. The hydrodynamic sizes of FeAC, CD, and CD-FeAC NPs averaged ~202/~337, ~10.5/~20.61, and ~362.3/~519.4 nm in PBS buffer/serum-free RPMI media at neutral pH, respectively. Generally in serum-free RPMI media, the hydrodynamic size was increased due to the strong ionic effects related to the aggregation behavior of NP colloidals. As a result, the NP's aggregation was induced. In comparison with TEM imaging analysis, DLS data showed relatively less aggregates. It may be related to drying effect for TEM sample preparation [41,45].

Cytotoxicity results of FeAC, CD, and CD-FeAC NPs
Based on the characterization data for FeAC, CD, and CD-FeAC NPs, MTT cytotoxic assay [29,49] was tested according to the sample loading concentrations (Fig. 5ac). FeAC NPs resulted in negligible cytotoxicity in normal cells but a slight (20 %) cytotoxic effect in cancer cells up to 1000 μg/mL. CD showed no cytotoxicity up to 1000 μg/mL. In CD conjugated FeAC (CD-FeAC) NPs, it was also reduced cytotoxicity due to biocompatible CD property and decreasing accessibility with Fe source in FeAC NPs. To see FeAC NPs cytotoxicity in detail, FeAC NPs were evaluated for other cancer and normal cell lines (Fig. 6), it resulted in slightly cytotoxic effects by reduced cell viability (%). Beyond mitochondria-based MTT assay, chromatin-based NR assays of FeAC, CD, and CD-FeAC according the loading concentrations in HeLa cells showed similar trends (Fig. 7).

NPs's observation of cellular uptake in HeLa cells
Remarkably, the cytotoxicity of CD-FeAC NPs in HeLa cells was slightly decreased at <1000 μg/mL, owing to the biocompatible CD conjugation, compared to that of FeAC treatment. Confocal microscopy images of the uptaken CD-FeAC NPs in HeLa cells and RAG cells showed a clear blue emission (Fig. 8a; Additional file 1: Figure S3a) in comparison with photos of fresh HeLa cells (Fig. 8b) and RAG cells (Additional file 1: Figure  S3b). Because FeAC NPs without fluorescent materials were discerned by contrast in cross-sectioned TEM image, the intracelluar location of only FeAC clusters in cross-sectioned HeLa cells was confirmed as the cytoplasm (Fig. 8c), indicating that the uptaken FeAC NPs showed no acute cytotoxicity to the living cell's morphology. Furthermore, it was confirmed by elemental mapping of the uptaken FeAC NPs into a single HeLa  (Fig. 9). In addition, confocal microscopy images of DAPI stained nucleus in the absence and presence of FeAC NPs were observed (Fig. 10). Only DAPI stained HeLa cells showed slightly scattered blue emission in nucleus sites (Fig. 10a) but clear blue emission in the presence of FeAC NPs showed spherical or elliptical morphology (Fig. 10b-e), interestingly, FeAC NPs may show a negligible change while nucleus staining, retaining nuclear integrity.

Discussion
The cytotoxic results of Mg-and Ca-aminoclays were researched as non-toxic nanomaterial for bio-medical and bio-imaging [29][30][31]. In this study, cytotoxicity of FeAC resulted in slightly cytotoxic effects by reduced cell viability (%), due to the oxidation of H 2 O 2 in cancer cells as a result of coordinated Fe 3+ source to hydroxyl free radicals (•OH) like Fenton-like reaction [50][51][52]. These •OH induced death in the cancer cell or apoptosis in vitro due to the radical-induced DNA-and cell-membrane damage. CD conjugation why it reduced cytotoxicity in FeAC NPs may be related to decreasing probability in accessibility of Fe source with cells. In detail, the reduced toxicity may be related to CD blocked the entrance or contact sites of FeAC for reactive oxygen species (ROS) generation, rather than the negatively charged surface property in CD-FeAC NPs.
In generally, anticancer agents delivered into cytosol and then, reached nucleus and directly effected in cell death [34]. So, tracking studies of CD-FeAC or FeAC NPs carriers into cells is important. As shown in Fig. 8c, FeAC NPs were existed into nucleus neighbors in HeLa cells abundantly, indicating CD-FeAC or FeAC NPs carriers can be successfully delivered target compounds into cytosol or nucleus in cells. In addition, nucleus site was negligibly damaged by FeAC NPs (Fig. 10). It is indicated that intact FeAC NPS have little cytotoxic effects for nucleus sites as well as an ability of transfection carriers [53][54][55]. As therapeutic agents, thus, taking into consideration the practical concentration in this study, <500 μg/ mL concentration of CD-FeAC is suitable to development of transfection reagent [29,31]. In conclusion, CD-FeAC NPs can be a useful diagnostic and therapeutic agent in providing drug-delivery-carrier with simultaneous fluorescent bio-imaging and tracking functionalities, although the use of only FeAC NPs can be given up the simultaneous bio-imaging.

Conclusions
In summary, it has dealt that CD-FeAC NPs plays roles both in bio-imaging and as a drug-delivery carrier into human cells with little cytotoxicity as simple preparation and inexpensive sources. In close future, cinnamic acid derivatives as anticancer agent can be loaded for target cargos [56] by amide bonding between carboxylic groups in cinnamic acid derivatives and amine groups in FeAC NPs. Taking into consideration that, as noted above, the practical applied dosage of NPs is <500 μg/mL, CD-FeAC NPs is feasible for trapping of targeting drugs and proteins, because it shows cytotoxicity only to cancer cells. Conclusively, a CD-FeAC-based hybrid agent for imaging/selective anticancer platform in in vivo is currently in the planning stage.

Preparation of Fe-aminoclay (FeAC) NPs
Synthesis of FeAC NPs was carried out according to the method available in the literature [40][41][42]. To a 500 mLbeaker solution containing 200 mL of ethanol, 8.4 g (31.08 mmol) of FeCl 3 •6H 2 O salt (Sigma-Aldrich, USA) was added. After complete dissolution by 10 min magnetic stirring, 13.0 mL (58.73 mmol) of 3-aminopropyltriethoxysilane (APTES, Sigma-Aldrich, USA) was added to the ferric (Fe 3+ ) ethanolic solution. In the course of mixing preparatory to the sol-gel reaction, brown slurry was formed. After 6 h equilibrium in production, the FeAC product was collected by 10 min 6000×g centrifugation. Then, after two ethanol-washing steps, the FeAC product was oven-dried at 50 °C for 1 day. Finally, preparatory to its use, the FeAC product was ground by pestle and mortar into a brown powder.

Preparation of carbon nanodots (CD) NPs
Organic waste solution (100 g of dried animal feces's wastes per 10 L of three double distilled water) at 45 °C was treated by 40 kHz ultrasound for 90 min (Ultrasonics UC-05, Lab Companion, Korea) [33,34]. It was then centrifuged at 2500 rpm for 5 min to remove large or agglomerated particles. The CD-containing supernatant was filtered twice through a 0.22 μm pore sized

Conjugation of CDs with FeAC (CD-FeAC) NPs
For conjugation on the surface of FeAC NPs, CD NPs  [31,40]. The final product (10 mg/mL) was re-suspended in PBS buffer and stored at 4 °C until use.

Characterizations of FeAC, CD, and CD-FeAC NPs
Transmission electron microscopy (FE-TEM, Tecnai TF30 ST, FEI company, USA) images were examined. Samples had been prepared for TEM imaging by dropping a tiny pipetted amount on a carbon-coated Cu grid (300-mesh) and oven-drying at 50 °C. After 2 h bathsonication of 2.0 mg/mL of FeAC and CD-FeAC NPs in PBS buffer and serum-free Roswell Park Memorial Institute (RPMI) media, respectively, their hydrodynamic diameter sizes and zeta potential values were measured using particle size analyzer (Zetasizer nano zs, Malvern, UK). For CD's height and surface roughness, after 100 μL of the CD solution was placed on a silicon wafer and air-dried overnight, it was scanned by atomic force microscope (AFM, VEECO Instrument, USA). Disorderinduced D and first-order graphite G bands in samples were recorded by Raman microscopy system (NT-MDT NTEGRA Systems, USA), utilizing photoluminescence (PL) spectroscopy with a changeable UV transilluminator (DUT-260, Core Bio Systems, Korea) within the 290-410 nm excitation range. In order to check intrinsic property of quantum-like structure of CD, the quantum yield of pristine CD at 3 mg/mL was examined by spectrofluorometer (FP-8500, Jasco, Japan) following the literature [33].

Cytotoxic evaluations of by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR) assays
In order to examine cytotoxic effect of samples, two assays such as mitochondria-MTT and chromatinbased NR means were approached. The designed cell line systems were organized with cancer and normal cells ( Table 1)

Cellular uptake observation by confocal microscopy and cross-sectioned transmission electron microscopy (TEM)
HeLa cells and RAG cells were cultured on an 8-well chamber slide with a concentration of 2 × 10 4 cells per well and on a 6-well chamber slide with a concentration of 2 × 10 5 cells per well for confocal microscopy (LSM510 META NLO, Carl Zeiss, Germany) and field emission transmission electron microscopy (FE-TEM, Tecnai TF30 ST, FEI company, USA), respectively. After 24 h incubation, cells were exposed to FeAC or CD-FeAC NPs for a further 24 h, and were then washed several times with PBS buffer. After drying the PBS on the 8-well chamber slide, the slide was covered with cover glass using fluorescent mounting media (DaKo). The fluorescence of CD-FeAC NPs in cells was measured under LSM510 META non-linear optic (NLO) confocal microscopy at excitation 345 nm and emission 460 nm wavelength. Cells in the 6-well plate were collected using trypsin-EDTA and washed twice with PBS buffer. Another observation of the nucleus in the FeAC NPs-treated and absence of FeAC NPs with HeLa cells, 4′,6-diamidino-2-phenylindole (DAPI, 1 μg/mL, 10 minincubation) staining protocol was followed and observed under confocal microscopy. As for the cross-sectioned TEM imaging, it followed the procedure available in the literature [45]. In detail, the FeAC NPs-treated HeLa cells were fixed in a 2.5 % paraformaldehyde-glutaraldehyde mixture buffered with phosphate (0.01 M and pH 7.2) for 2 h, post-fixed in 1.0 % osmium tetroxide in the same buffer for 1 h, dehydrated in graded ethanol and propylene oxide (PPO), and embedded in Epon-812. Ultra-thin sections, cut by the ULTRACUT E (Leica, Austria) ultramicrotome, were stained with uranyl acetate and lead citrate and examined under CM 20 electron microscopy (Philips, Netherlands).

Additional file
Additional file 1: Figure S1. Raman spectra of FeAC, CD, and CD-FeAC NPs. Figure S2. Height roughness image (a) with including height (nm) analysis (b) of CD NPs by atomic force microscope (AFM). Note that in (a) the black underlining marks were employed as size guides. Figure S3.