Synthesis and Characterization of Human Skin-Compatible Conductive Flexible Cellulose Carbon Nanohorn Sheets

Background: Conductive sheets of cellulose and carbon nanomaterials and its human skin applications are an interesting research aspect as they have potential for applications for skin compatibility. Hence it is needed to explore the effects and shed light on these applications. Method: To fabricate wearable, portable, exible, lightweight, inexpensive, and biocompatible composite materials, carbon nanohorns (CNHs) and hydroxyethylcellulose (HEC) were used as precursors to prepare CNH-HEC (Cnh-cel) composite sheets. Cnh-cel sheets were prepared with different loading concentrations of CNHs (10, 20 50,100 mg) in 200 mg cellulose. To fabricate the bio-compatible sheets, a pristine composite of CNHs and HEC was prepared without any pretreatment of the materials. Results: The obtained sheets are conductive (1.83×10 -5 S) and bio-compatible with human skin. Analysis for skin-compatibility was performed for Cnh-cel sheets by h-CLAT in vitro skin sensitization tests to evaluate the activation of THP-1 cells. It was found that THP-1 cells were not activated by Cnh-cel; hence Cnh-cel is a safe biomaterial for human skin. It was also found that the composite allowed only a maximum loading of 100 mg to retain the consistent geometry of free-standing sheets of < 100 µm thickness. Since CNHs have a unique arrangement of aggregates (dahlia structure), the composite is homogeneous, as veried by transmission electron microscopy (TEM) and, scanning electron microscopy (SEM), and other functional properties investigated by Raman spectroscopy, Fourier transform infrared spectroscopy (FT-IR), conductance measurement, tensile strength measurement, and skin sensitization. Conclusion: It can be concluded that cellulose and CNHs sheets are conductive and compatible to human skin applications.


Introduction
Among all the nanomaterials, carbon nanomaterials exhibit the most unique properties. Many prominent applications involving materials synthesized with carbon nanomaterials have gained popularity in recent years. Owing to their unique geometry, and properties closely related to single-walled carbon nanotubes (SWCNTs), carbon nanohorns (CNHs) have attracted signi cant attention. However, CNHs have different pro les of dispersion, structural geometry, surface chemistry, and synthesis methods and hence have a different properties. The toxicity of carbon nanomaterials is also a signi cant limitation for bio-related applications. Many metals are used as catalysts for the synthesis of carbon nanotubes (CNT) and other carbon nanomaterials (1). Owing to the inter-wall attraction force (van der Waals) of the CNTs, the dispersion of CNTs is a cumbersome process, and harsh chemical treatments using acids, and surfactants are necessary (2,3).
CNHs may be an effective alternative to CNTs because they exhibit excellent purity and does not require post-synthesis treatment. CNHs have horn-shaped capped ends of approximately 2-3 nm, the wider ends aggregate to other CNHs, resulting in a Dahlia-like spherical structure (4). The void between the tips of adjacent CNHs allows the CNH aggregates to have an active dispersion pro le compared to CNTs.
Although functionalization improved CNHs dispersion, the CNH aggregates could not be separated into single CNHs by chemical functionalization (5). The three-dimensional arrangement of the CNHs aggregates allows electron transport and in uences the bonding within the polymer along the surface, enhancing the mechanical properties of the composite, compared to the one-dimensional con nement of CNTs. Research on oxidized CNHs to entrap anticancer agents to treat lung cancer was reported as early as 2005 (6). Polymer composites comprising poly-(vinyl alcohol), graphene, and CNHs have also been reported recently (7).
On the other hand, cellulose is an abundantly available natural polymer (8). In particular, Hydroxyethylcellulose (HEC) is a derived cellulose that is used as a thickening agent, which are widely used materials in industries. HEC is also known for its application in the pharmaceutical industry for capsule formulations to improve hydrophilization of drugs (9). Research on cellulose composites with carbon nanomaterials has been increasingly, with multiple reports on the numerous applications of cellulose-carbon nanomaterials. CNTs and cellulose composites have recently been studied as aerogels for vapor sensing (10) and, water sensors (11). In addition, graphene oxide coated with cellulose nano bers have been used for designing transparent conductive paper (12). A few other have reports included the doping of conductive nanomaterials such as Ag nanowires, for electromagnetic interference shielding (13) and, PEDOT: PSS/MWCNT for supercapacitor electrodes (14).
In the area of smart, bio-compatible, wearable devices carbon nanomaterials are being widely researched. To accommodate a device in close contact with human body/skin the compatibility of the material is a very crucial aspect. To evaluate allergic reactions and safety of different materials to human skin, will help in further exploring different applications of new materials. Such as, allergic dermatitis caused by skin sensitizers is one of the prominent items safety compliance considerations. To evaluate the skin sensitization potential of the prepared Cnh-cel sheets, h-CLAT, an in vitro skin sensitization test [including cytotoxicity test] was carried out. The cytotoxicity of CNH is expected to be low because of its spherical aggregates, as no metal catalyst is used during the synthesis of CNHs. CNHs and their composite materials are applicable to living bodies, especially in the medical eld, safety evaluations, such as those evaluating toxicity and allergy, are strictly tested and regulated for safety.
For the safety evaluation of substances, allergic dermatitis is one of the essential items. After skin contact, substances causing allergic reactions, such as rash, are termed skin sensitizers, and the processes causing allergic reactions are termed skin sensitization (GHS 2017). The mechanisms of skin sensitization have been summarized in the form of an adverse outcome pathway (AOP) ranging from early events at the molecular level to adverse events through intermediate events, including the following four events (OECD 2014) (16). The rst event is the formation of covalent bonds between the electrophile; in other words, skin sensitizers covalently bind to the nucleophilic center of the proteins present in the skin. The second event includes in ammatory reactions, particularly in the keratinocytes in the skin, activation of the antioxidants/electrophilic substance responsive element (AREs) dependent signal transduction pathways. The third event is the activation of antigen-presenting cells called dendritic cells in the immune system, which is assessed by the expression of speci c cell surface markers, chemokines, and cytokines. The fourth event is the proliferation of T cells, which play a central role in the host immune response. The evaluation of skin sensitization using animal (in vivo) and non-animal (in vitro) experiments aims to reproduce all or part of the AOP. In vivo animal experiments include guinea pig maximization (OECD 1992) (17) and the mouse regional lymph node test (OECD 2010) (18).
From the viewpoint of animal protection, regulations on animal experiments have become stricter in recent years. Particularly in the cosmetic industry in the EU, animal experiments for the production and evaluation of raw materials, processed products, and nal products, have been banned (Ban on animal testing 2019). Additionally, sales and imports of such materials and products have also been banned; therefore, manufacturers exporting these to the EU are forced to comply. Thus The h-CLAT is a test method to evaluate the third event of the AOP by measuring the expression level of the cell surface marker (OCED, 2016) (30). It is known that upon the activation of dendritic cells, the expression levels of cell surface markers such as CD86 and CD54 increase also (31). In h-CLAT, the human monocytic leukemia cell line THP-1 cells were used as the dendritic cell model to evaluate the ability of test substances to activate dendritic cells.
In this study, we aimed to evaluate skin sensitization and cytotoxicity of novel composites of CNH and cellulose sheets using h-CLAT. These Cnh-cel sheets enabled the application of the novel composites in the eld of restricted animal experiments.

Cnh-cel sheet synthesis
Hydroxyethylcellulose, carbon nanohorns, and deionized water were used for cnh-cel sheet synthesis. A standard solution of HEC was prepared by dissolving 200 mg of cellulose in DI water. The cellulose nano bers, when dissolved, can untangle or dissociate by subjecting them to ultra-sonication at a frequency of 23 kHz for 30 min. The cellulose solution was heated at 85 °C for 30 min to completely dissolve the cellulose bers, which resulted in a transparent solution. CNHs were dispersed in DI water at loading concentrations of 10 mg, 20 mg, 50 mg, and 100 mg. The CNH solution was sonicated for 1 h. Initially, the CNHs precipitated due to their agglomeration and dahlia-like structures. When subjected to low-frequency ultra-sonication, an immediate dispersion was observed. The ratio of CNH to DI water was maintained at a very high rate, such that CNH exhibited enough room to disperse from their aggregated dahlia-like structures. Both solutions were divided into different portions depending on the ratios of HEC:CNH and then sonicated for 30 min to produce a volatile solution of different ratios of HEC:CNH which was agitated thoroughly. The combined solution was heated with stirring initially at 80 °C for 4 h at 200 rpm; to evaporate excess DI water, and the viscosity of the volatile solution started to increase.
Subsequently, no excess DI water could be evaporated from the concentrated solution; the heat was raised to 90 °C for an hour and then nally to 100 °C. This resulted in a highly viscous, solution. The nal solution was poured into a syringe, and a few droplets of this solution were dropped onto a clean glass plate to draw uniform sheets using the leveling applicator (bar coater). The sheets were allowed to dry at room temperature, and trapped a few molecules of water to maintain the structure. After a few minutes, the sheets were peeled off from the glass plate and analyzed using TEM, SEM, Raman, FTIR, as well as tensile strength, and conductance measurements, and the skin sensitization test. A schematic of the graphical representation of the Cnh-cel sheet, and its conductive path is shown in Fig. 1.
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).

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 nal 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 × 10 6 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 ow cytometry, and the concentration (CV75) of the test sample in 75% cell viability was determined using Formula 1.

In vitro skin sensitization test
The results of the cytotoxicity and test dose determination tests are shown in Fig. 2. The CV 75 value was determined using Formula 1.
Cell viability was measured using ow 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 ow cytometry. RFI was calculated using MFI. The black bar represents the rst 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 Fig. 6. 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).

Transmission Electron Microscopy
To investigate the interparticle interaction between the CNH aggregates, a transmission electron microscope was employed. Pristine carbon nanohorn samples were prepared by sonicating pristine samples in ethanol and DI water, and drop-casting on a copper grid.
Similarly, cellulose samples doped with CNHs were diluted with DI water and drop-cast on to the copper grid and dried on a hot plate for a few minutes. As shown in the TEM images in Figs. 7(a) and 7(b), the pristine samples of CNH have cone-like structures, with single and double-walled CNHs arranged in dahlia-like structures, with the conical tip prodding out of the dahlia-like assembly. The pristine CNHs have cone tips of 2-3 nm in thickness. Figure 7(c) shows that these dahlia-like structures are accompanied by a layer of cellulose coating on the outer side connecting adjacent dahlia-like structured CNHs. This shows that the cellulose has adhered both in between and on top of the conical tips of the CNHs. As shown in Fig. 7(d), a distorted ring-like structure of amorphous carbon surrounding the dahlialike structures, can be seen when magni ed at 50 nm using TEM.

Scanning electron microscopy
Scanning electron microscopy of the Cnh-cel sheets is shown in Figs. 8(a) -8(d). The Cnh-cel sheets are observed under SEM at an accelerating voltage of 5 kV. The morphology of the Cnh-cel sheets determine various parameters about the Cnh-cel sheets.

Raman spectroscopy
Raman spectroscopy characterization is a useful method of analysis to evaluate the purity of carbon nanomaterials. As interpreted from the plot in Fig. 9, CNHs exhibit two bands at 1,338 cm − 1 (D band) and 1,580 cm − 1 (G-band) (32).

Tensile strength measurement
Tensile strength measurement is a crucial component in determining the durability and strength of the sheets. 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 Fig. 11. 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.
A typical measurement of conductance for sheets is determined by its area of cross-sectional area and length. However, the conductance varies with the variation in the length and thickness of sheets. By keeping the parameters of length and thickness to a certain roundoff, the conductance is determined. The conductance measurements are proportional to the level of doping of CNH in the cellulose matrix as the CNH doping increases the conductance of the sample by 100%. However, we could not determine the maximum doping possible to retain the geometry of the thin lm for < 100 µm sized sheets at 50 wt% cellulose. Figure 13 shows the combined plot of the voltage (V) vs. current (I), however, the current values are different for all the Cnh-cel sheets.

Discussion
The present report on Cnh-cel composite sheets and its test for skin compatibility test is the rst report and there is not history of any previous reports for this study available according to our literature survey.
In vitro skin sensitization test the concentration of Cnh-cel composites and cell viability were inversely proportional (Fig. 3). 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.
In the transmission electron microscopy, the interaction between the long chains of cellulose nano bers and the carbon nanohorns can be observed in the TEM images. Therefore, it can be concluded that even without any surface modi cation 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 con rmed by FTIR analysis.It is seen in the SEM images that, as the CNH loading concentration increase, the smoothness of the surface of the Cnh-cel sheets changes gradually. With the 100 mg doping concentration of CNH shown in Fig. 8(d), the surface morphology of the Cnh-cel sheet is rough and porous compared to the 10 mg doping, as shown in Fig. 8 (a). In addition, as in Fig. 8 (b) and 8 (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 con rmed using the tensile strength measurement. In conclusion, the magni ed images of the surface of the Cnh-cel sheets show that the strength and conductance are inversely related to the concentration of the cellulose and CNHs.
Raman spectroscopy The D band is observed because of the sp 3 carbon, which produces elastic scattering. The intensities of I d and I g correspond to the sp 3 and sp 2 hybridization of carbon atoms present in the CNHs (I d /I g = 1.2). For pristine CNH, the D band is signi cantly higher than that of 10 mg CNH-doped sheet. The presence of D and G bands con rm the presence of CNHs in the Cnh-cel sheets. The increase in the Raman intensities corresponds to the increase in the CNH loading concentration but not in exact proportion to the loading concentration. In the Fourier transform Infrared spectroscopy, it is a well-known fact that the cellulose is a hygroscopic material, hence few bonds are related to the hygroscopic property, and OH − bonds are detected at 3,340 cm − 1 also involving weak 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 identi ed at 1,562 cm − 1 .
In the tensile strength of Cnh-cel sheets, 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 Fig. 11. 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 lms 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 (33) 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 Fig. 12. In the conductance measurement, the conductance 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 conductance 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 conductance of 6.145 × 10 − 6 S for 50 mg loading and 1.88 × 10 − 5 S for 100 mg loading.

Conclusion
Cellulose carbon nanohorn composite sheets (Cnh-cel sheets) with different loading concentrations were successfully fabricated in our study. The properties of the Cnh-cel sheets were analyzed and determined using various characterization techniques. The conductance of the Cnh-cel sheet at the maximum loading concentration was measured to be 1.88 × 10 − 5 S, and the skin application of the Cnh-cel sheets to human skin was also tested, and it was proven to be safe for applications on human skin. These initial experiments of Cnh-cel sheets shed light on the understanding of carbon allotropes application as smart materials. The encouraging properties of Cnh-cel sheets such as exibility, strength, conductive, and skincompatible indicate their high potential for various modern-day applications.   The application of Cnh-cel sheets on human skin and passage an electric current to light up LEDs Raman plot of CNH-cel sheets  Tensile strength measurement of the Cnh-cell sheets Figure 12 Cnh-cel sheet tensile measurement setup