In vivo guiding nitrogen-doped carbon nanozyme for tumor catalytic therapy

Nanomaterials with intrinsic enzyme-like activities (nanozymes), have been widely used as artificial enzymes in biomedicine. However, how to control their in vivo performance in a target cell is still challenging. Here we report a strategy to coordinate nanozymes to target tumor cells and selectively perform their activity to destruct tumors. We develop a nanozyme using nitrogen-doped porous carbon nanospheres which possess four enzyme-like activities (oxidase, peroxidase, catalase and superoxide dismutase) responsible for reactive oxygen species regulation. We then introduce ferritin to guide nitrogen-doped porous carbon nanospheres into lysosomes and boost reactive oxygen species generation in a tumor-specific manner, resulting in significant tumor regression in human tumor xenograft mice models. Together, our study provides evidence that nitrogen-doped porous carbon nanospheres are powerful nanozymes capable of regulating intracellular reactive oxygen species, and ferritinylation is a promising strategy to render nanozymes to target tumor cells for in vivo tumor catalytic therapy.


Supplementary Discussion
As shown in Supplementary Fig. 3a, X-ray diffraction patterns displayed two peaks near 24 o and 42 o , presenting the characteristics for the (002) and (100) graphitic planes. The formation of the graphitic structure was further identified using Raman spectroscopy (Supplementary Fig. 3b). The results showed an intense and narrow band at 1590 cm -1 (G-band, the first-order scattering of the E2g vibrations observed for sp 2 domains in an ideal graphitic layer) and 1340 cm -1 (D-band, correlated to structural defects and partially disordered structure of sp 2 domains). The peak intensity ratios of D and G bands (I D /I G ) for PCNSs, N-PCNSs-5 and N-PCNSs-3 were measured as 0.934, 0.925 and 0.926, respectively. These data indicated that the obtained carbon materials have been highly graphitized. XPS results showed that N-PCNSs-5 and N-PCNSs-3 were N-doped to 2.85 atom% and 3.37 atom%, respectively, while the PCNSs showed no nitrogen content (Supplementary Fig. 3c and Supplementary Table 1).
The bonding configurations of the nitrogen atoms of N-PCNSs were characterized by high resolution N 1s spectra. There were four peaks at 398.38 eV, 399.78 eV, 401.18 eV, and 402.88 eV, presenting pyridinic nitrogen (N-6), pyrrolic nitrogen (N-5), quaternary nitrogen (N-Q) and pyridine oxide or the oxidized nitrogen (N-O X ), respectively. Compared to N-PCNSs-3, the N-6, N-Q and N-O X configurations except N-5 in N-PCNSs-5 were decreased (Supplementary Table  2). This indicated that prolonging carbonization time at high temperature affected the amount and type of N-doping in carbon materials. The high-resolution O 1s spectrum of the N-PCNSs-3 revealed that the O atoms mainly were in three types, C=O (530.98 eV), C-OH (532.58 eV) and COOH (534.28 eV) (Fig. 1e), implying that oxygen-containing functional groups were present on the surface of the material.
The C 1s high resolution XPS spectrum of PCNSs ( Supplementary Fig. 3d) exhibited four main peaks at 284.6, 285.1, 286.3, and 288.0 eV, which were assigned to C-C, C-O, C=O and O-C=O, respectively. In N-PCNSs-5 and N-PCNSs-3, the intensity of the C-O/C-N peaks was increased (Supplementary Figs. 3e and 3f), further indicating that N was doped into the carbon framework.
The FTIR spectrum of PCNSs showed that plenty of O functional groups, such as O-H and C=O, presented on the surface of PCNSs, indicating that hydroxyl or carboxyl groups were formed on the surface of PCNSs ( Supplementary Fig.4). With the N-doping, we found that the characteristic absorption bands of N-H were stretching vibrations at 3770 cm -1 , as well as aromatic C-N stretching vibration at 1234 cm -1 . Moreover, the C=O bands located at 1712 cm -1 were also observed in N-PCNSs, and the band intensity was decreased with the prolong carbonation time in N-PCNSs-5 compared to N-PCNSs-3.
As shown in Supplementary Fig. 5, BET Isotherm was performed to get further insight about surface area and porous structure. The surface areas of PCNSs, N-PCNSs-3 and N-PCNSs-5 were determined as 583.8，542.1 and 614.8 m 2 g -1 , respectively. The pore sizes of PCNSs, N-PCNSs-3 and N-PCNSs-5 were determined as 2.2, 3.0 and 3.1 nm, respectively. These data indicated that addition of melamine did not cause obvious difference in surface area and porous structure of PCNSs.