A Sub-Nanostructural Transformable Nanozyme for Tumor Photocatalytic Therapy

Highlights An internal sub-nanostructural transformation of sub-nanostructural transformable gold@ceria (STGC-PEG) is initiated by the conversion between CeO2 and electron-rich state of CeO2−x, and active oxygen vacancies generation via the hot-electron injection from gold to ceria. The sub-nanostructural transformation of STGC-PEG enhances the peroxidase-like activity and activates the plasmon-promoted oxidase-like activity, resulting in an augmented reactive oxygen species output. STGC-PEG successfully achieves excellent low power (50 mW cm−2) near-infrared light-activated photocatalytic ablation of tumors in vivo. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-022-00848-y.

In living systems, the regulation of catalytic activities of natural enzymes generally depends on the dynamic rearrangement of their intrinsic structures [23][24][25]. For instance, chymotrypsinogen can be activated by trypsin-mediated cleavage of peptide bonds between Arg15 and Ile16 [26], and DNA photolyase would undergo a concerted structural change during photoactivation to initiate the repair of the impaired duplex [27]. The structural reconfiguration-medicated regulation of enzymatic activities is essential to the coordination of numerous biochemical events in living systems [24,28,29], which may represent the ultimate and yet to be implemented strategy to endow nanozymes with natural enzyme-like regulatability.
Inspired by nature, we herein propose a photon-driven sub-nanostructural transformable nanozyme that performs tunable catalytic activities via intrinsic sub-nanostructural transformation. Plasmonic metal nanomaterials (e.g., Au, Ag nanostructures) have a unique surface plasmon resonance (SPR) effect and can generate energetic hot electrons upon resonant light excitation [30][31][32][33]. Particularly, when coupled with semiconducting nanomaterials (e.g., CeO 2 , TiO 2 nanostructures), the electronic properties of the conjoined nanomaterials can be further exploited through the direct plasmon-excited electron transfer, thus boosting their photocatalytic performances [34][35][36]. Consequently, the designed integration of plasmonic metal/semiconductor nanostructures is anticipated to achieve the photon-driven sub-nanostructural transformation via regulating their electronic properties to initiate the local atomic reconstruction [37,38], so as to facilitate the catalytic activity regulation of nanozymes.
To prove our concept, a sub-nanostructural transformable gold@ceria (STGC) nanozyme was synthesized by controlled assembly of ultrafine ceria nanoparticles (CeO 2 NPs) onto the plasmonic gold nanorods (GNRs), which provides strong near-infrared (NIR) light absorption and abundant surface reactive sites for efficient catalytic reactions [39]. Unprecedentedly, once triggered by 808 nm irradiation, plasmon-excited hot electrons directly transferred from Au to CeO 2 , converting CeO 2 to electronrich state of CeO 2-x and inducing the generation of active oxygen vacancies (OVs) to dynamically reconstruct the 1 3 sub-nanostructure of STGC-PEG (Fig. 1a). Such an internal sub-nanostructural transformation ingeniously regulated the peroxidase (POD)-and oxidase (OXD)-like catalytic activities of STGC-PEG for reactive oxygen species (ROS) generation, enabling highly efficient photocatalytic therapy (PCT) of tumors. The as-designed sub-nanostructural transformable STGC-PEG represents a proof-of-concept of natural enzyme-like catalytic activity regulation  Fig. 1 Design and characterization of photon-driven sub-nanostructural transformable nanozymes. a Schematic illustration of the design and photon-driven sub-nanostructural transformation of STGC through the direct electron transfer. Once triggered by near-infrared (NIR) irradiation, plasmon-excited hot electrons directly transfer from Au to CeO 2 , thus converting CeO 2 to electron-rich state of CeO 2−x , and producing active oxygen vacancies (OVs) to dynamically reconstruct the sub-nanostructure of STGC. b TEM image of STGC. Scale bar, 100 nm. c HAADF-STEM image of a single STGC and the corresponding elemental mapping. Scale bar, 20 nm. d HRTEM images of STGC. Scale bar, 10 nm. e XRD pattern of STGC. f UV-Vis absorption of GNRs and STGC. The LSPR peak of GNRs red shifts from 781 to 807 nm after CeO 2 coating. g TEM image of PEGylated STGC (STGC-PEG). Inset: hydrodynamic diameter distribution of STGC-PEG. Scale bar, 100 nm. h, i Cyclic voltammograms of STGC (black line) and STGC + laser (red line, 808 nm) without (h) and with H 2 O 2 treatment (i). j Raman spectra of STGC after different treatments (L denotes laser) (λ ex = 488 nm). k, l HAADF-STEM images of STGC treated with H 2 O 2 before (k) and after (l) 808 nm laser irradiation. The O-related defect induced lattice disorder is indicated by the dashed circles. Scale bar, 1 nm Data are presented as means ± s.d. (n = 3/group). g ESR spectra of DMPO-OH spin adducts generated by STGC-PEG with or without low-power 808 nm laser irradiation (50 mW cm −2 , 5 min) in the presence of H 2 O 2 (10 mM). h, i ESR spectra of TEMP-1 O 2 (h), and BMPO-˙O 2 − (i) spin adducts generated by STGC-PEG with or without low-power 808 nm laser irradiation (50 mW cm −2 , 5 min). j Schematic illustration of atomiclevel photon-mediated sub-nanostructural transformation of STGC-PEG for enzymatic activity regulation. Upon NIR irradiation, the generated hot electrons from Au can convert CeO 2 to CeO 2−x and generate active OVs, thus endowing STGC-PEG with photon-promoted peroxidase (POD)-and oxidase (OXD)-like activities 1 3 via allosteric nanozymes for precisely controllable ROSbased nanomedicines.

Synthesis of STGC
The CTAB-capped GNRs were synthesized by the typical seed-mediated method [40]. NaBH 4 (10 mM, 50 µL) was injected into a mixture containing CTAB (0.1 M, 1.95 mL) and HAuCl 4 (10 mM, 50 µL) and stirred for 2 min to obtain the seed solution, which was placed at 27 °C for 2 h before use. For the preparation of growth solution, HAuCl 4 (10 mM, 15 mL), AgNO 3 (4 mM, 7.5 mL), AA (0.1 M, 1.2 mL) and the seed solution (450 µL) were injected into CTAB (0.1 M, 300 mL), and the solution was stirred for 2 min and then kept undisturbed at 27 °C at least 3 h. The final product was collected by centrifugation at 10,000 rpm for 15 min and re-dispersed into deionized water (DI water) as the stock solution (0.4 nM). STGC was synthesized through a modified solvothermal method [39]. For the preparation of STGC, CTAB (0.2 M, 1.125 mL), EDTA-NH 3 (10 mM, 0.35 mL) and CeCl 3 (0.1 M, 0.035 mL) were sequentially added into the GNRs stock solution (8 mL). Then, the solution was kept at 90 °C for 1.5 h in an oven. The final product was centrifuged and re-dispersed into DI water (2 mL) for further use. 5K (mPEG 5K -ALN) mPEG 5K -ALN was synthesized according to the reported method by Yang et al. [41]. Briefly, mPEG 5K -COOH (500 mg), EDC (30 mg), and NHS (15 mg) were dissolved into DI water (4 mL) and stirred for 30 min. Then, alendronate sodium trihydrate (100 mg) and Na 2 CO 3 (40 mg) were dissolved in DI water (2 mL) and added to the above solution. The reaction was carried out for 24 h, followed by dialysis and freeze-drying before collecting the final product.

Surface Modification of STGC
STGC (70 μg mL −1 , 2 mL), mPEG 5K -SH (10 mg), and mPEG 5K -ALN (30 mg) were mixed in 2 mL DI water for the ligand exchange process, and the mixture was stirred at room temperature (RT) for 24 h. The resulting PEGylated STGC was isolated via centrifugation and rinsed three times with DI water for further use.

Synthesis of STGC
Ultrafine CeO 2 NPs were assembled on the surface of GNRs (Fig. S1) through a modified solvothermal method [39] to form well-dispersed STGC (Figs. 1b and S2-S3). Energydispersive spectrum (EDS) (Fig. S4a), line-scanning EDS (Fig. S4b), and elemental mapping (Fig. 1c) illustrate the distribution of Ce and O on the surface of GNRs. The mass ratio of Au: Ce in STGC is ~ 3: 1 as measured via inductively coupled plasma mass spectrometry (ICP-MS), where the relatively high Au-to-Ce ratio guarantees the sufficient hot electrons supply from Au and further photon-driven sub-nanostructural transformation of STGC. High-resolution transmission electron microscopy (HRTEM) (Fig. 1d) and X-ray diffraction (XRD) (Fig. 1e) pattern reveal the (111) plane of the fluorite cubic CeO 2 phase and the (111) plane of cubic Au phase, demonstrating the poly-crystalline nature of STGC. Besides, the red-shift of longitudinal surface plasmon resonance (LSPR) peak further suggests the successful coating of CeO 2 on GNRs, owning to the CeO 2 coating-induced local increase of refractive index, where strong NIR adsorption endows STGC with potential phototriggered catalytic activity [39] (Fig. 1f). Moreover, X-ray photoelectron spectroscopy (XPS) spectrum reveals the coexistence of Ce 3+ and Ce 4+ in STGC (Fig. S5), suggesting the presence of OVs that maintain the charge balance [5,38]. For further biomedical applications, the STGC was modified with poly(ethylene glycol) (PEG) (Fig. 1g), and the PEGylated STGC (STGC-PEG) with an average hydrodynamic diameter of ~ 128.5 nm (Fig. 1g, inset) and a negative charge (Fig. S6), is highly stable in water (Fig. S7). The successful PEGylation was further confirmed by XPS and Fourier transform infrared (FT-IR) (Fig. S8).

Photon-Driven Electron Transfer-Mediated Sub-Nanostructural Transformation
To verify the photon-driven electron transfer-mediated sub-nanostructural transformation of STGC, cyclic voltammetry (CV) curves of STGC were initially recorded. The symmetrical redox peaks at around + 0.51 and + 0.35 V are assigned to the redox of Ce 3+ /Ce 4+ in CeO 2 [42,43]; upon NIR irradiation, a significant peak current enhancement of STGC can be observed (Fig. 1h). The electric current enhancement agrees well with the proposal that the plasmon-excited hot electrons overcome the Schottky barrier and then transfer from Au to CeO 2 [39,44], leading to the generation of Ce 3+ and active OVs [42,45,46]. Intriguingly, the increase in electric current also occurred in H 2 O 2 -pretreated STGC (Fig. 1i). Besides, the regeneration of Ce 3+ and OVs of CeO 2 NPs in the presence of H 2 O 2 would be inhibited in acidic conditions [38,47]. Therefore, the regeneration ability of CeO 2 NPs in STGC can be restored, benefiting from the electron transfer from Au to CeO 2 upon laser irradiation. Consistently, the LSPR peak of STGC red-shifts (Ce 3+ → Ce 4+ ) after co-incubation with H 2 O 2 in acidic condition [39] and returns upon laser irradiation (Fig. S9). Moreover, as shown in Raman spectra (Fig. 1j), the initial 455 cm −1 peak (a symmetric breathing mode of oxygen atoms surrounding Ce ions [47,48]) diminishes and a new peak at ~ 837 cm −1 (O-O stretching vibration of the absorbed peroxide species [49]) is observed after co-incubation with H 2 O 2 in acidic condition, which can also be recovered after laser (L) irradiation. Furthermore, XPS and electron spin resonance (ESR) results manifest the formation of OVs in H 2 O 2 -treated STGC after laser irradiation ( Fig. S10a-b), while the XRD patterns show that the crystal planes of CeO 2 (JCPDS No. 034-0394) do not reveal a significant change after reaction, indicating the light-mediated tunable catalytic activities are not based on the change of crystal planes (Fig.  S10c). Impressively, more lattice disorder and dislocation upon laser irradiation are directly verified for H 2 O 2 -treated STGC by using atomic-resolution high-angle annular darkfield scanning transmission electron microscopy (HAADF-STEM) (Fig. 1k, l), which indicates the existence of numerous vacancies for the induction of coordinatively unsaturated metal atoms [50,51], demonstrating the photon-driven subnanostructural transformation of STGC.

Low-Power NIR Light-Activated Tumor PCT
The photocatalytic effect of STGC-PEG was further investigated at the cellular level. STGC-PEG can be effectively internalized by 4T1 tumor cells (Figs. S15 and S16) without inducing noticeable dark cytotoxicity (Fig. 3a). Moreover, despite that GNRs-PEG show a photothermal conversion efficiency (η, 39.7%) comparable to that of STGC-PEG (37.2%; Fig. S17), only STGC-PEG shows significant cytotoxicity upon low-power NIR irradiation (50 mW cm −2 ), which decreases the cellular viability to 59.7% at the concentration of 25 μg mL −1 Au (Fig. 3a, b), with the most severe ROS generation (Fig. 3c), mitochondrial damage (Fig. 3d) and apoptosis rate of tumor cells (Fig. 3e). Besides, STGC-PEG displays a much lower cytotoxicity on human normal cell lines including hepatic cell L02 and colonic epithelial cell NCM460 even upon NIR irradiation, probably resulting from the relatively low level of H 2 O 2 in normal cells (Fig.  S18). Taken together, we conclude that upon internalization into tumor cells, the conversion from CeO 2 to electron-rich state of CeO 2−x as well as the active OVs generation would be initiated by the hot-electron injection from gold to ceria upon low-power NIR irradiation, whereafter the dynamic sub-nanostructural transformation of STGC-PEG triggers the excess ROS generation for effective tumor cell killing. Furthermore, we examined the changes in the molecular pathway of 4T1 tumor cells after different treatments. As shown in Fig. 3f, the levels of Akt, phosphorylated Akt (p-Akt), p-PI3K and p-FoxO, which are well implicated in breast cancer development, are downregulated by STGC-PEG upon NIR irradiation. Moreover, STGC-PEG induces the downregulation of antiapoptotic Bcl-xl, the upregulation of proapoptotic Bax and cleaved caspase-3 (cl-caspase-3) during the PCT. Consequently, we reason that the toxic ROS generated by STGC-PEG not only directly affect Bcl-2 family proteins and activate pro-apoptotic pathways [64], but also inhibit the PI3K/Akt/FoxO signaling cascade, thus promoting the tumor cell death (Fig. 3g).
Encouraged by in vitro results, we further evaluated the in vivo tumor PCT using STGC-PEG under low-power NIR irradiation (50 mW cm −2 ). Considering that high light fluency can inevitably induce the necrosis of the normal tissues [65,66], it is appealing to achieve potent phototherapy with minimal light intensity, especially for deep-seated tumors requiring light penetration through tissue barriers that diminish light intensity at the tumor sites. Due to the power attenuation in thick biological tissues, the power density of laser sharply declines as measured by a power meter, for example, by 85.1% and 92.2% at tissue thickness of 5 mm and 7 mm, respectively ( Fig. 4a-d). Interestingly, because of the sharp NIR light attenuation in deep tumor tissues, through hematoxylin and eosin (H&E) staining of the whole tumor after the laser irradiation, we find that MGC-PEG leads to limited tumor necrosis due to the power-dependent photothermal effect, while STGC-PEG induces severe tumor necrosis with a much larger area and greater depth (Fig. 4e). This finding strengthens the advantage of the low-power NIR irradiation-triggered deep tumor PCT by using STGC-PEG via the markedly high-performance photon-mediated sub-nanostructural transformation.
Consequently, STGC-PEG effectively suppresses the progression of 4T1 breast cancer, while MGC-PEG shows no obvious tumor growth inhibition (Figs. 4f-g and S19). Furthermore, the ROS level in STGC-PEG + laser-treated tumor presents to be the highest among all the groups, indicating the highly efficient in situ photocatalytic reaction. H&E and terminal deoxynucleotidyl transferase dUTP nick end label (TUNEL) staining results reveal not only significantly severe cell apoptosis, but also markedly deep tumor destruction in STGC-PEG + L group (Fig. 4h). Notably, similar to control group, neither obvious body weight change (Fig. S20), nor noticeable pathological tissue damage or abnormality from the histology analysis (H&E staining) (Fig. S21) can be found in mice treated with STGC-PEG. Besides, body weights (Fig.  S22), serum biochemical analysis (Fig. S23), hematological index (Fig. S24), as well as H&E staining (Fig. S25) of healthy normal BALB/c mice all demonstrate the great biocompatibility of STGC-PEG in vivo. To our best knowledge, this is the first demonstration of a nanozyme that, by virtue of the dynamic sub-nanostructural transformation-mediated catalytic activity regulation, can achieve excellent low-power NIR lightactivated photocatalytic ablation of tumors in vivo; such a subnanostructural transformable nanozyme enabled tumor PCT is superior to conventional light-mediated therapies, such as PTT and photodynamic therapy, whose efficacy can be significantly affected by light attenuation in tissues or tumor hypoxia.

Conclusions
We have developed a photon-driven sub-nanostructural transformable nanozyme (STGC-PEG) with light-responsive sub-nanostructural transformation that can fine-tune its catalytic activities in biological environment. Upon NIR irradiation, the plasmon-excited hot electrons transfer from Au to CeO 2 to initiate CeO 2 -CeO 2−x conversion and generate active OVs, endowing STGC-PEG with dramatically amplified POD-and OXD-like activities. Importantly, STGC-PEG results in massive augmentation of ROS production with spatiotemporal controllability upon low-power NIR irradiation and thus potentiate the anti-tumor efficiency of PCT both in vitro and in vivo. Particularly, for the first time, STGC-PEG successfully achieves excellent low power NIR light-activated photocatalytic ablation of tumors in vivo. As a proof-of-concept, our strategy provides a new paradigm for fabricating dynamically transformable nanozyme with tunable catalytic performances in vivo for biomedical applications. Our findings may aid the future design of advanced biomimetic nanocatalysts and provide a model for approaching natural enzyme-like activity control of artificial nanozymes.