{'fraction_non_alphanumeric': 0.03663455997365605, 'fraction_numerical': 0.022639334815180705, 'mean_word_length': 5.110663983903421, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Atomically Precise Water‐Soluble Graphene Quantum Dot for Cancer Sonodynamic Therapy (Adv. Sci. 19/2022)

Graphene Quantum Dots An atomically‐precise graphene quantum dot with bilayer stacking (structures shown in cover picture) is synthesized and monodispersed in water owing to introducing sterically hindered water‐soluble functional groups. Under the ultrasonic irradiation, it can generate abundant cellular reactive oxygen species (detected as the green fluorescence of cells shown as the background of the cover picture), and thus shows excellent tumor inhibition potency. More details can be found in article number 2105034 by Gang Liu, Yuan‐Zhi Tan, and co‐workers.


Introduction
Graphene quantum dots (GQDs), as a class of zero-dimensional flat graphenic materials with lateral sizes smaller than 10 nm. Water-soluble GQDs have been proven to be potential in various biological applications, [1] such as bioimaging, sensing, and biological therapy. However, the large heterogeneity in compositions, sizes, and shapes of these water-soluble GQDs results in significant challenges in better understanding their structureproperties correlation and further clinical uses in terms of large-scale manufacturing practices and safety concerns. On the other hand, GQDs with clear and precise structures can be synthesized by rational organic route using dehydrocyclization of dendritic polyphenylenes. [2] These welldefined organically-synthesized GQDs have shown applications in electronics, optics, and energy conversion [3] but are rarely used in biology, [4] because most of them are naturally water-insoluble and prone to aggregate into stacked large particles due to strong interactions between the nano-scale planar carbon skeleton. A few well-defined warped GQDs functionalized by oligoethylene-glycol chains can be water-soluble, due to the hindered stacking between their warped skeleton, [4a,5] however, they were still inclined to assemble into nano-sized aggregations. [5] A general synthetic strategy to make these atomically-precise GQDs soluble and monodispersed in water is highly demanded, which can provide a series of well-defined nanomaterials beyond the small molecules for bio-applications, for example, sonodynamic therapy (SDT).
porphyrin compounds, xanthone products, and antitumor drugs, among others. However, traditional molecular sonosensitizers exhibit relatively low ROS production efficiencies. Meanwhile, traditional molecular sonosensitizers suffer from ROS-mediated self-destruction, leading to chemical instability under US conditions. [12] Nanomaterial-based sonosensitizers, [13] exhibit good stability and enhanced ROS generation. For example, the carbon nanomaterials could have a long excitation lifetime [14] and multistate sensitization, [15] that could enhance the ROS generation. However, despite the advantages of nanomaterial sonosensitizers in terms of potency, most lack well-defined structures. The development of new sonosensitizers combining well-defined structures of small molecules with superior sonosensitization efficacies of nanomaterials is crucial for the development of SDT.
Here we show a water-soluble atomically precise GQD, namely WAGQD-C 96 , was prepared by introducing sterically hindered water-soluble functional groups. The well-defined chemical and assembly structures of WAGQD-C 96 were characterized by mass, NMR, and UV-vis spectroscopies. The high-resolution transmission electron microscopy (HRTEM) and dynamic light scatter-ing (DLS) confirmed the monodispersion of WAGQD-C 96 in water without further aggregation. The biological behaviors of WAGQD-C 96 , including cellular uptake, cellular SDT effects, and retention in tumor, were probed by its deep-red emission. WAGQD-C 96 was proven to be able of generating more ROS under US irradiation than typical organic molecular sonosensitizers. In vivo studies indicated WAGQD-C 96 can greatly inhibit tumor growth in terms of SDT without obvious toxicity.
Similar to previously reported C 96 GQD functionalized with mesityl groups, [14] compound 2 also adopted a discrete bilayer structure without further intermolecularstacking. This was validated by mass, NMR, and UV-vis spectroscopies (Figures S8-S10, Supporting Information). The formation of discrete bilayer structure is due to the balance between the stereo-hindrance of peripheral substituents and stronginteractions of inner core. [16] Next, the hydrolysis of the ester groups in substituents led to the formation of 4-carboxy-2,6-dimethylphenyl functionalized C 96 GQD (1) (Figure 1a). The mass spectrum of 1 showed a dominant peak at 4144.108 Da, confirming the preservation of the well-defined bilayer structure after hydrolysis of ester groups ( Figure 1b). Then, compound 1 was dissolved in water containing an equivalent of potassium carbonate, to produce the WAGQD-C 96 aqueous solution. The WAGQD-C 96 aqueous solution (pH = 7.3) was used for further photophysical and bio-application studies.
As shown in Figure 2a, the UV-vis spectrum of an aqueous solution of WAGQD-C 96 exhibited absorption bands at 463 and 528 nm, assigned to p and absorption bands. WAGQD-C 96 revealed a deep red fluorescence (FL) with three peaks at 650, 694, and 760 nm, among which the second peak at 694 nm showed the highest intensity (Figure 2a). Such spectral features confirmed the H-aggregated bilayer structure of WAGQD-C 96 without intermolecularstacking under aqueous conditions. A typical Raman spectrum and IR spectrum (Figures S11 and S12, Supporting Information) confirmed the sp 2 carbon skeleton of WAGQD-C 96 . The morphology of WAGQD-C 96 in an aqueous solution was studied by HRTEM. As shown in Figure 2b, abundant uniform particles with a size of 1.6 ± 0.2 nm were observed (Figure 2b). Note that the particle size matched well with the diameter of graphenic core (1.8 nm) for a single WAGQD-C 96 (Figure 2b). Thus, particles observed by HRTEM imaging could be assigned to a single WAGQD-C 96 molecule, suggesting the monodispersed WAGQD-C 96 in water, in consistence with spectral characterizations. DLS analysis of WAGQD-C 96 aqueous solution showed a narrow size distribution (PDI = 0.21) with an average hydrodynamic radius (R h ) of 1.5 nm (Figure 2c and Figure  S13, Supporting Information), validating the monodispersion of WAGQD-C 96 in aqueous solution.

Fluorescent Imaging by WAGQD-C 96
The water-solubility of WAGQD-C 96 enables the studies on its biological behaviors, which can be conveniently probed by the deep-red emission of WAGQD-C 96 . In cellular level assays, confocal laser scanning microscopy (CLSM) was carried out and monitored, showing that WAGQD-C 96 presented time-dependent cel- lular uptake (Figure 3a). The flow cytometry analysis showed the similar cellular uptake of WAGQD-C 96 ( Figure S14, Supporting Information). The deep-red emission of WAGQD-C 96 can be also used for fluorescent diagnosis and visualization of tumors. As depicted in Figure 3b and Figure S15, Supporting Information, the FL images and calculated mean intensities indicated good photoemission and concentration-dependent relationship of WAGQD-C 96 . The time-dependent retention of WAGQD-C 96 in the tumor region was then visually inspected after intratumoral injection. Long-time retention of WAGQD-C 96 in tumor tissue was observed with signals mostly detained up to 72 h (Figure 3c and Figure S16, Supporting Information). All animal assays were carried out deferring to the protocols approved by Xiamen University Laboratory Animal Center (XMU2021101).

SDT Using WAGQD-C 96
Besides its deep-red photo-emission, the US-responsive ROS sensitization capability of WAGQD-C 96 was evaluated using 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) as ROS probe. Various concentrations of WAGQD-C 96 ( Figure S17, Supporting Information) generated increased ROS levels under ultrasound irradiation at 0.56 W cm −2 for 5 min. Compared to the reported well-defined organic sonosensitizers like P18, meso-tetrakis (4-sulfonatophenyl) porphyrin (TPPS), indocyanine green (ICG), and porphyrin, WAGQD-C 96 exhibited superior performance toward the production of ROS (Figure 4a). Meanwhile, sonosensitizers must ensure low dark cytotoxicity as well. A safe concentration of WAGQD-C 96 was determined by MTT assays as 20 μm ( Figure S18, Supporting Information). Then the cellular level SDT effect of WAGQD-C 96 was evaluated. As shown in Figure 4b and Figure S19, Supporting Information, abundant cellular ROS after 6 h internalization was observed under ultrasound irradiation at 0.56 W cm −2 for 5 min. In lysosome escape results (Figure 4c), the US treatment was able of destroying lysosomes and promoting its escape due to ROS generation. This would be conducive to the toxic generation of ROS in cytoplasm, useful for directly attacking the nucleus. [17] MTT assays were used to evaluate the SDT effect of WAGQD-C 96 . Compared to WAGQD-C 96 without ultrasound treatment ( Figure S20, Supporting Information), the cell viability of HepG2 cells significantly decreased with WAGQD-C 96 concentration after ultrasound treatment for 5 min at 0.56 W cm −2 . A fairly low IC50 of 9.8 μm manifested a high SDT efficiency of WAGQD-C 96 .
Encouraged by the excellent ROS performance and obvious SDT effect at the cellular level, the feasibility of WAGQD-C 96 as SDT reagent for antitumor was investigated in vivo. In view of the long-term retention of WAGQD-C 96 , we exploited two-injection and four-ultrasound irradiation for antitumor therapy. To this end, the volumes and images of tumors and body weights of four groups were recorded every two days during 12 consecutive days of therapy and observation period. As shown in Figure 5a,b, no obvious tumor outburst was recorded after initial ultrasound treatment of WAGQD-C 96 + US group. By contrast, the tumor volumes in other control groups increased sharply, indicating that SDT induced by WAGQD-C 96 could dramatically eliminate the tumor. Also, WAGQD-C 96 + US groups displayed a superior tumor inhibitory rate of 70 % when compared to the other three groups ( Figure S21, Supporting Information).
The anatomized tumor of WAGQD-C 96 under US irradiation also revealed the lowest values in both size and weight (Figure 5c,d) than other control groups. Next, pathological analyzes by H&E and TUNEL staining were performed to evaluate the ex vivo tumor tissues. As shown in Figure 5e, high tumor elimination and tumor cell apoptosis, including cell shrinkage and fragmentized nuclei were observed in WAGQD-C 96 + US group. Meanwhile, the body weights remained steady during the initial day, suggesting negligible physical toxicity of WAGQD-C 96 (Figure S22, Supporting Information). The ex vivo analysis of major organs, including the heart, liver, spleen, lung, and kidney, revealed no distinct damage, further confirming the biosafety of WAGQD-C 96 ( Figure S23, Supporting Information). The hemanalysis also confirmed no obvious biotoxicity of WAGQD-C 96 during in vivo tests ( Figure S24, Supporting Information).

Conclusion
In summary, a simple and general strategy was developed to obtain WAGQD by decorating sterically hindered water-soluble functional groups on the well-defined GQD core, which can address the long-standing issue on the synthesis of monodispersed water-soluble GQDs. WAGQD-C 96 here was characterized as a bilayer structure and can be monodispersed in water without further stacking, showing a well-defined chemical and assembled structure. Probed by its deep-red emission, good cellular uptake and long-time retention in tumor were discovered. Both in vitro and in vivo experiments demonstrated the superior ROS sensitizing potency of WAGQD-C 96 under US irradiation coupled with good biosafety. Consequentially, WAGQD-C 96 can greatly inhibit tumor growth in in vivo SDT. Combined with the general synthetic strategy of WAGQD, other WAGQDs with different sizes, edges, and tailored properties can be developed for effective SDT, as well as for other bio-applications.

Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.