3 Results and discussion
The novel RCNs were synthesized by soft-template hydrothermal method. As indicated in the TEM image (Figure 1 ), it was apparently demonstrated the products were constituted principally by rod-shaped carbon particles with relatively uniform dispersion and regular particle size. And the average size of the carbon rods was measured to be approximately 85-120nm in length, and 35-50nm in width. This novel morphology might be originated from the rodlike glucose-F127 monomicelles form, in which the polymerization, dehydration and nucleation of glucose took place, and finally form the rod-shaped nano structures.
The hydrodynamic diameter of RCNs was then measured by DLS. As shown inFigure S1, the size of the particles was calculated to be 110nm, which was consistent with the TEM results. Furthermore, the stability test of the RCNs was characterized by DLS over 2 weeks, as shown inFigure S2 . It could be seen that the size exhibited almost negligible changes, which proved that the RCNs possess good stability in aqueous solution system.
The surface functional groups of carbon nanomaterials were further analyzed by FT-IR. Figure 2 showed that the absorption peak at 3600-3200cm-1 was the stretching vibration of hydroxyl O-H, the absorption peak at 3000-2800cm-1 was the stretching vibration of saturated C-H, the absorption peak at 1770-1680cm-1 was the stretching vibration of ester carbonyl C=O, and the absorption peak at 1250-1000cm-1was the stretching vibration of ester C-O.
The UV-vis-NIR spectra of the obtained RCNs in aqueous solution was shown in Figure 3 , which revealed that the samples possessed high absorption in the NIR region around 650-900nm. The excellent NIR absorption characteristic of the samples encouraged us to explore their photothermal properties. The RCNs aqueous solution with different concentrations were irradiated under an 808nm laser at 2.5W/cm2 power density. As shown in theFigure 4 , under 808nm laser, the water temperature only increased by 3°C within 5 min. And it could be seen that the temperature of the RCNs solution increased more and more rapidly as the concentration increased. When the concentration was 50μg/mL, the temperature could raise to 45℃ in 5 min, while the concentration was 100μg/mL, it could raise to 54℃. The photothermal conversion efficiency of RCNs was calculated to be about 23.7 % using the reported literature. In the course of 6 cycles of photothermal heating and natural cooling (Figure S3 ), the photothermal heating effect did not change significantly, indicating that the RCNs had good photothermal stability.
In order to improve the therapeutic effect of the nanomaterials toward tumors, the nanocomposite with integrated thermo-chemotherapy performance was fabricated, while the classic DOX was used as the model anti-tumor drug. By the analysis of the DOX@RCNs, the drug loading rate of DOX for the nanocomposites was calculated to be 17.4%. The photothermal heating test was further arranged for the DOX@RCNs at the concentration of 50μg/mL. It could be seen in the Figure 5 that the temperature could raise to 39℃ in 5 min, and 52℃ in 10 min, which showed excellent photothermal heating effect.
The drug release experiment under 808 nm laser radiation was explored, it could be seen from Figure 6 that the release rate of DOX in the composites could achieve at 34.6% in 60 min, after 2h, the rates was calculated to be 64.7%. In the comparative experiments without irradiation, the corresponding releasing rates were calculated to be 5.7% and 7.2%, respectively. It could be concluded that infrared radiation can significantly increase the release of DOX, further realize integrated treatment of hyperthermia and chemotherapy.
Considering that low toxicity is a necessary condition for nano-drugs, the cell viability of MCF-7 cells incubated with different concentrations of RCNs and DOX@RCNs were conducted. The cell viability of MCF-7 cells incubated with two novel nano-drugs was shown inFigure 7 . It could be observed that the carbon nanomaterials exhibit excellent biocompatibility, and have little cytotoxicity to MCF-7 cells up to a concentration of 300μg/mL. For comparison, the cytotoxicity for DOX@RCNs was slightly stronger. The cell survival rate decreased gradually with the increasing of the concentration of the sample, when the concentration was 300μg/mL, the cell survival rate was 78.6%.
Both the RCNs and DOX@RCNs were further explored their potential antitumor activity under NIR irradiation against MCF-7 cells. It could be seen that there was almost no cytotoxicity at the concentration of 50μg/mL for both RCNs and DOX@RCNs without NIR irradiation (Figure 7 ). In contrast, under NIR irradiation, two novel nano-drugs exhibited enhanced cytotoxicity against MCF-7 cells (Figure S4 ). It should be noted that no obvious cell apoptosis occurred after the MCF-7 cells were irradiated in the culture medium without the nanomaterials. The relative cell viabilities were calculated to be 50.2% at the RCNs concentration of 50μg/mL after 5 min of continuous irradiation (2.5W/cm2), suggesting the excellent photothermal toxicity effect of the RCNs. For the DOX@RCNs samples, benefiting from the effective NIR-sensitive release of DOX, DOX@RCNs exhibited stronger cytotoxicity than that of RCNs, while the relative cell viabilities decreased to 23.6% at the same test conditions.
In addition, confocal fluorescence microscopy was used to explore the toxicity effects of the nanomaterials (Figure 8) . DAPI, which could strongly bind to DNA, is a blue fluorescent dye used for fluorescence microscopy observation of living and fixed cells. In addition, fluorescein-dUTP is a green fluorescent dye which is used to label apoptotic cells. In this research, in order to display the cell apoptosis promoted by nanomaterials, DAPI/fluorescein-dUTP assays were performed to intuitively observe live and apoptotic cells. After 4 min irradiation, it could be seen that obvious apoptosis appeared in the MCF-7 cells for the RCNs group compared with the control ones, however, much more extensively apoptosis occurs for the DOX@RCNs group. This phenomenon was consistent with the MTT results.