In the TEM images the interaction between the long chains of cellulose nanofibers and the carbon nanohorns can be observed. Therefore, it can be concluded that even without any surface modification of the CNHs, interactions between CNHs and cellulose can be achieved. It is also observed that each dahlia-like aggregate of CNHs has a uniformly layered covering of cellulose; showing all the virtuous composites of cellulose and CNHs. This covering of cellulose to the CNH aggregates makes the CNHs aggregate as core-shell type nanoparticles, which can also be used in other applications where dissociation of the CNHs is not necessary and the dahlia-like structure is kept intact while allowing the CNHs to interact with other molecules via the cellulose shell. The interaction between the CNHs and cellulose can also be confirmed by FTIR analysis.
As per the SEM images the 100 mg doping concentration of CNH shown in Figure 3(d), the surface morphology of the Cnh-cel sheet is rough and porous compared to the 10 mg doping, as shown in Figure 3(a). In addition, as in figure 3(b) and 3(c), the CNH doping concentrations are 20 mg and 50 mg, respectively, and the surface morphology is greatly varied. This indicates that the cellulose concentration in the Cnh-cel sheets is responsible for the surface smoothness and strength, as confirmed using the tensile strength measurement. In conclusion, the magnified images of the surface of the Cnh-cel sheets show that the strength and conductivity are inversely related to the concentration of the cellulose and CNHs.
In the Raman analysis the D band is observed because of the sp3 carbon, which produces elastic scattering. The intensities of Id and Ig correspond to the sp3 and sp2 hybridization of carbon atoms present in the CNHs (Id/Ig= 1.2). When pristine CNH is compared to the other loading concentrations of CNH with cellulose the Id band intensity is reduced gradually as the loading concentration is decreased. The elastic scattering of Id band is affected significantly more that Ig band indicating the increase in amorphous carbon content in the cnh-cel sheets. Similarly it is a well-known fact that the cellulose is a hygroscopic material, hence few bonds are related to the hygroscopic property are expected in FT-IR analysis, and OH- bonds are detected at 3,340 cm-1 also involving weak C-H stretching at 2,896 cm-1, both of which explain the hygroscopic nature of cellulose. Furthermore, an absorption band at 1,065 cm-1 corresponding to the –(COH) functional group was also observed, and also a COO functional group is was identified at 1,562 cm-1. The intensities of different loading concentrations are represented by the intensity peaks in Figure 5. As the doping concentration of CNH increases from 10 mg to 100 mg the intensity of –(COH) functional group is decreased. Also, OH- peak is affected by the increase in the doping of CNH which could explain the tensile strength decreasing of the cnh-cel sheets by increasing the loading concentration.
In the Tensile measurement it is observed that, as the CNH loading increases, the strength decreases as the CNHs loading reduces the interparticle bonding between the adjacent cellulose molecules. The CNH loading concentration of 10 mg had a displacement of 3.074 mm having an ultimate strength to withstand a force of 1001.50 mN, as shown in Figure 6. In contrast, the 20 mg loading was able to withstand a displacement of 1.62 mm but had an ultimate strength to tolerate a force of 990.7 mN until they broke. This shows that when the loading concentration is doubled, and the displacement was reduced by 52.7%, but the ultimate strength was only affected by 9.8%. Similarly, at 50 mg loading, the displacement was 1.2 mm at which it could stand 865.79 mN of force, whereas for 100 mg loading the displacement was 0.23 mm for the ultimate strength to bear the force of 164.24 mN. The threshold of the force endurable for Cnh-cel sheets for pristine Cnh-cel composite are collectively plotted in CNH loading versus force, and the displacement is shown below in Figure 6. The plot shows a trend of a decrease by the force and displacement proportional to the loading concentration, which enables us to understand the suitable loading concentrations based on the type of application. It was found that a further increase in the loading concentration of CNHs could not provide structural integrity to fabricate thin films of less than 100 µm in thickness, which could be related to the upper limit of doping, possibly for pristine CNHs to cellulose ratios. However, functionalization of the CNHs and dissolving the cellulose in ionic liquids (38) could increase the endurance of Cnh-cel composite sheets, while compromising other properties. A typical setup of the tensile strength measurement of a Cnh-cel sheet is shown in Figure 7.
The conductivity measurements are proportional to the level of doping of CNH in the cellulose matrix as the CNH doping increases the conductivity of the sample by 100%. However, we could not determine the maximum doping possible to retain the geometry of the thin film for < 100 µm sized sheets at 50 wt% cellulose. Figure 8 shows the combined plot of the voltage (V) vs. current (I), however, the current values are different for all the Cnh-cel sheets. The conductivity of the sheets with 10 mg and 20 mg loading of CNHs show a non-linear V-I curve, which indicates that the sheets are not good conductors, and the conductivity cannot be determined as the sheets do not have a linear V-I curve. However, at 50 and 100 mg loading of CNHs, a linear V-I curve is observed, which translates to a conductivity of 6.145 × 10-11 S/m for 50 mg loading and 1.83×10-10 S/m for 100 mg loading.
The skin sensitization test method is outlined as follows:
This study was conducted according to the OECD Guidelines for the Testing of Chemicals (OCED, 2016).
THP-1 cells (RCB1189, Riken, RBC, Tsukuba, Japan) were cultured in an incubator (37 °C and 5% CO2) using RPMI-1640 medium (Nacalai Tesuque, Kyoto, Japan) supplemented with 10% fetal bovine serum (FBS) (Nichirei Biosciences, Tokyo, Japan), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin in 100 µg/mL streptomycin (both Nacalai Tesuque). The cells were seeded at a density of 0.1-0.2 × 106 cells/ mL once every 2 to 3 days to maintain a density of 0.1 - 1.0 × 106 cells/mL.
Determination of test dose
Three doses of working solutions were prepared as follows. Cnh-cel composites were added to the cell culture medium at a final concentration of 8,666 µg/mL. This working solution was serially diluted at 1:2 to prepare the remaining two working solutions. Eighty microliters of the cell suspension at a density of 2 × 106 cells/mL was seeded into each well of a 96-well plate, and the cells were added with 80 µL working solutions or the medium (negative control) and cultured in an incubator for 24 h. Cells were then collected into 1.5 mL tubes and centrifuged at 250 × g, 4 °C, for 5 min. Cells were resuspended in 500 µL of FACS buffer (PBS supplemented with 0.1% BSA). After 10 washes with repetitive resuspension and centrifugation, the cells were re-suspended in 200 µL FACS buffer. Immediately before measurement, the cells were stained by adding 5 µL propidium iodide (PI) (Invitrogen, Carlsbad, CA) solution (20 µg/mL diluted with FACS buffer). Cell viability was measured using flow cytometry, and the concentration (CV75) of the test sample in 75% cell viability was determined using Formula 1.
See formula 1 in the supplementary files.
Skin sensitization test
Four doses of Cnh-cel composite working solutions at concentrations of CV75 × 1.2-2, -1, 0, 1 were prepared. Cells were seeded, working solutions added, cultured, and washed as described above. After centrifugation, the cells were blocked with 330 µL of blocking solution (FACS buffer containing 0.01% human globulin Cohn fraction II, III (Sigma-Aldrich, St. Louis, MO)) for 15 min at 4 °C. The cells were divided into three tubes with 100 µl and added 30 µl of diluted anti-CD86 (BD-PharMingen, Franklin Lakes, NJ), anti-CD54 or mouse IgG 1 (both Dako, Santa Clara, CA) antibodies. To make the diluted antibody solutions, the antibodies and FACS buffer were mixed at the ratios between antibody solutions and FACS buffer were at 6:44, 3:47, and 3:47, respectively. After 30 min at 4 °C, cells were washed with FACS buffer twice and re-suspended in 150 µL of FACS buffer followed by the addition of 4 µL of the diluted PI solution. The relative fluorescence intensity (RFI) was calculated using Formula 2 based on the measured mean fluorescence intensity (MFI) obtained by flow cytometry, as an indicator of CD86 and CD54 relative expression.
See formula 2 in the supplementary files.
In vitro skin sensitization test
To evaluate the Cnh-cel sheets in skin sensitization test, the CV75, the does whose cell viability is 75%, defined in Formula 1 was determined as 1,999 µg/mL using experimentally obtained parameters, a = 89.5%, b = 1,083 µ/mL, c = 73.1 %, d = 2,166 µg/mL. The test doses, CV75 * 1.2^(1, 0, -1, and -2) were then calculated as 2399 µg/mL (CV75 × 1.2), 1999 µg/mL (CV75), 1,666 µg/mL (CV75/1.2), 1,388 µg/mL (CV75/1.22), respectively. The results of the cytotoxicity and test dose determination tests are shown in Figure 9. The CV 75 value was determined using Formula 1.
In this test, a = 89.5%, b = 1,083 µ/mL, c = 73.1 %, d = 2,166 µg/mL. The CV75 value of Cnh-cel composites was 1,999 µg/mL. The exposure concentration of the Cnh-cel composites was set at four levels, diluted at a ratio of 1-2 based on CV75. The value were 2399 µg/mL (CV75 × 1.2), 1999 µg/mL (CV75), 1,666 µg/mL (CV75/1.2), 1,388 µg/mL (CV75/1.22).
Cell viability was also confirmed microscopically shown in Figure 9. Note that the Cnh-cel composites appeared to be a little toxic at the higher concentrations shown in Figure 10. However, the CV75 of Cnh-cel composites was much higher than that of lactic acid, which is a standard negative control in this test (1000µg/mL) suggesting that the cytotoxicity of the Cnh-cell composites is very low. The concentration of Cnh-cel composites and cell viability were inversely proportional (Figure 10). Thus, a high concentration of Cnh-cel composites is cytotoxic. The CV75 value of the Cnh-cel composite was 1999 µg/mg. The CV75 value of lactic acid, which is used in cosmetics suggests a relatively low cytotoxicity value of 1000 µg/mL. Given the comparison of the CV75 values, the cytotoxicity of the Cnh-cel composites is relatively low.
Aliquots of cells were taken from the assays and imaged (a) just before starting the assay, (b) after washing cells and centrifugation, (c) 24 h after cells incubated with negative control, (d) 24 h after cells incubated with Cnh-cell composites and centrifuged. media control (b) Media control (incubate 24 h) (c) Media control (after centrifugation) (d) Cnh-Cell sheets (after centrifugation)
The skin sensitization is defined as the RFI (the relative fluorescence intensity calculated by Formula 2) of CD86 ≥ 150 and CD54 ≥ 200. As shown in Figures 11 and 12, the RFIs of CD86 and CD54 were lower than 150 and 200, respectively, in all the tested cases indicating that the Cnh-cell composites are not skin sensitization agents. Note the assay was performed twice. Each is shown in the closed bars and open bars in Figures 5.4 and 5.5. The results of the skin sensitization tests are shown in Figures 11 and 12. The results demonstrate that the RFI values of CD86 and CD54 are lower than the positive criteria (CD86 RFI ≥ 150, CD54 RFI ≥ 200). Therefore, Cnh-cel composites were identified as skin sensitization-negative in this experiment.
THP-1 cells were incubated with Cnh-cel composites (three doses: 4,333 µg/mL, 2,166 µg/mL, 1,083 µg/mL) or medium (negative control, 0 µg/mL) for 24 h. The cells were stained with PI solution. Cell viability was measured using flow cytometry.
THP-1 cells were incubated with Cnh-cel composites (four doses) for 24 h. The cells were stained with anti-CD86, anti-CD54, or mouse IgG-1 antibodies. MFI was measured using flow cytometry. RFI was calculated using MFI. The black bar represents the first result, and the white bar represents the second result. The Cnh-cel composites treated group showed that RFI values of CD86 and CD54 are lower than the positive criteria (CD86 RFI ≥ 150 and CD54 RFI ≥ 200). A working example of Cnh-cel sheets attached to human skin with glowing LED lights is shown in Figure 13. Notably, these Cnh-cel sheets did not exhibit skin sensitization in other bioassays, such as OECD TG442C (Amino acid Derivative Reactivity Assay) or OECD TG442D (data included in Supplementary Information).