English

• Photothermal therapy (PTT), a non-invasive tumor treatment modality, has attracted increasing attention in recent years [1]. PTT relies on photothermal conversion agents (PTCAs) to convert the light energy into heat energy, which can generate heat and ablate tumor cells under laser irradiation [2,3]. In general, PTCAs contain gold nanoparticles [3,4], black phosphorus [5], carbon nanotubes [6-8] and other organic nanomaterials [9,10], which have excellent photothermal conversion efficiency in the near-infrared (NIR) window (650–1350 nm). Currently, there are two types of PTT, harsh PTT (hPTT) and mild PTT (mPTT), have been conceived as the major therapeutic modalities. The hPTT can induce tumor cell necrosis by elevating local temperature to over 50 ℃, which tends to damage the surrounding normal tissues and the host anti-tumor immunity [11,12]. In contrast, mPTT induced antitumor effect through heat stress at a mild temperature (42–46 ℃) [13-15]. The mPTT cause tumor cells apoptosis by induce heat stress, which including protein and nucleic denaturation [16]. However, there are precisely defensive mechanisms to resist heat stress. Firstly, heat stress trigger heat shock response (HSR), in turn, up-regulate heat shock proteins to assist misfold and unfold proteins eliminating or recovery, leading to cell survival [17]. Secondly, autophagy is another procedure for impaired organelles and accumulated misfold and unfold proteins removing [18]. In addition, heat stress also triggers the endoplasmic reticulum (ER)-associated unfolded protein response (UPR), which help to maintain cellular homeostasis by increasing the protein folding capacity of ER [19]. These defensive mechanisms lead to thermoresistance, which seriously reduce the therapeutic efficacy of mPTT. In order to overcome thermoresistance, combination therapies are proposed, such as combining with chemotherapy [20-22].

  HSR is triggered by the hyperthermia-induced accumulation of misfolded or unfolded proteins to insure the protein homeostasis [17]. In this connection, heat shock proteins (HSPs) play a critical role in HSR [17]. Once heat treated, the up-regulated HSPs (e.g., HSP70, HSP90 and HSP110) would facilitate the elimination of the misfolded or unfolded proteins through ubiquitin-proteasome system and autophagy [23,24]. Hence, to target the HSPs will be an effective approach to improve the therapeutic efficacy of mPTT by avoid the thermoresistance [25-27]. Besides, HSPs was reported to be closely associated with multiple diseases, especially cancers [28-30].

  Histone deacetylase 6 (HDAC6), a unique histone deacetylase of the IIb HDAC family, has been implicated in the regulation process of mitosis, cell proliferation, invasion and metastasis in multiple cancers [31,32]. Interestingly, HDAC6 localizes primarily in the cytoplasma and also targets a variety of non-histone proteins substrates [33]. Notably, HSP90 is a key cytoplasm substrate of HDAC6 [34]. Under stressful condition (i.e., hyperthermia), HDAC6 up-regulates the HSP90 expression by activating HSF1 [35]. In addition, HDAC6 binds and deacetylates HSP90, which mediates the elimination of misfolded and unfold proteins by ubiquitin-proteasome system and autophagy [34]. Thus, HDAC6 inhibition is a potential strategy to overcome thermoresistance and increase the therapeutic efficacy of mPTT. Riconilostat (ACY-1215), a selective HDAC6 inhibitor with IC₅₀ of 4.7 nmol/L [36,37], was widely used in clinical trials for different cancers monotherapy or in combination with other treatments [32,33,38]. For instance, ACY-1215 treatment can activate caspase-3 and poly(ADP-ribose) polymerase (PARP), and induce apoptosis [37]. Furthermore, combined with bortezomib, ACY-1215 can inhibit aggresome formation and accelerate the cell death [39]. However, the combination of HDAC6 inhibitor and mPTT on tumor therapy has not been reported.

  Herein, to elevate mPTT efficacy for tumor therapy and antagonize mild PPT-induced thermoresistance (Scheme 1), an integrated nanoplatform (denoted as PMH) was designed and prepared, which contains a PdMo bimetallene with favourable photothermal effect and photoacoustic imaging capability, and a specific HDAC6 inhibitor (ACY-1215) (Fig. S1A in Supporting information). Firstly, the released ACY-1215 can inhibit HDAC6 to suppress mPTT-caused HSPs upregulation. Secondly, ACY-1215-induced hyperacety-lation of HSP90 promotes HSP90 dissociation with its client proteins, which maintain the stability and function of HSP90-client complex. These effects decrease the mPTT-induced misfold and unfold proteins degradation through proteasomal degradation. In addition, mPTT leads to autophagy system collapse by amplifying the ACY-1215-triggered autophagy process. Taken together, the combination of ACY-121 and mPTT reduces the two major ways for eliminating misfolded and unfold proteins, ubiquitin-proteasome system and autophagy, ultimately accelerating cell death.

  Scheme 1

  

  Scheme 1.  Schematic illustration of the preparation of PdMo bimetallene with loading of ACY-1215 and its application in enhanced mild photothermal therapy.

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  The PdMo bimetallene nanosheet (PdMo) was prepared by a one-pot wet-chemical approach [40]. The transmission electron microscopy (TEM) image shows that the PdMo is dominated by sheet-like morphology with an average diameter of 60 nm (Fig. 1A). The lattice spacing of 0.23 nm obtained from high-resolution transmission electron microscopy (HRTEM) image (Fig. 1B) corresponded well to the plane of the Pd (111) facet [41,42]. The power X-ray diffraction (XRD) pattern suggests that the PdMo possesses a face-centered cubic structure (Fig. 1C), in agreement with a previous report [43]. The high resolution X-ray photoelectron spectroscopy (XPS) spectra of (Fig. 1D) Pd 3d and Mo 3d (Fig. 1E) confirm that the Mo and Pd in bimetallene materials are mainly in their metallic state [43]. In order to the increase the stability and biocompatibility, the PdMo was modified with trithiol-terminated poly(methacrylic acid) (PTMP-PMAA) [40]. The ultraviolet–visible–near infrared (UV–vis–NIR) spectra (Fig. 1F) indicate that PdMo possesses an obvious absorption in both the NIR-I and NIR-II regions, which increases with increasing concentration of PdMo. Upon 1064-nm laser irradiation, the aqueous temperature significantly rose in a PdMo-induced, concentration-dependent, and laser power density-dependent manner (Figs. 1G and H), and PdMo had superior photothermal stability (Fig. 1I). Afterwards, the PdMo loaded with HDAC6 inhibitor (ACY-1215) was prepared for further study (Figs. S1B and C in Supporting information).

  Figure 1

  

  Figure 1.  Characterization of photophysical performance. (A) TEM image of PdMo bimetallene. (B) HRTEM image of PdMo bimetallene. (C) EDS elemental mapping of Pd and Mo in a single bimetallene nanosheet. (D) XRD spectra of PdMo bimetallene. High resolution XPS spectrum of I Pd 3d and Mo 3d for PdMo bimetallene. (F) UV–vis–NIR spectra of PdMo bimetalleneat different concentrations. (G) Concentration-dependent photothermal heating curves of PdMo bimetallene (0, 5, 10, 20, 40 µg/mL) under 1064 nm laser irradiation for 5 min. (H) Power density-dependent photothermal heating curves of PdMo bimetallene (0.2, 0.4, 0.6, 0.8, 1.0 W/cm²). (I) Photothermal stability of PdMo bimetallene (1064 nm, 0.6 W/cm²).

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  The in vitro therapeutic efficacy of PdMo plus HDAC6 inhibitor (ACY-1215) was evaluated. Mouse breast cancer cell (4T1) and human breast cancer cell (MDA-MB-231) lines were treated with PdMo or PMH with an increasing of concentrations (0, 5, 10, 20, 40 µg/mL). Under the laser irradiation, cell viability was analyzed at the time points of 24 and 36 h, respectively. As shown in Figs. 2A and B, without loading ACY-1215 and laser treatment, the cytotoxicity of PdMo was negligible even at 40 µg/mL. Treated with PMH or laser decreased cell viability. Significantly, loading ACY-1215 plus laser treatment could effectively inhibit cell viability at a low concentration at 5 µg/mL, indicating the synergistic effects. However, no effect of synergy was observed at high dose level (40 µg/mL). This could be explained that the high dose of ACY-1215 loaded is sufficient to inhibit the cell viability. Similar results were observed in MDA-MB-231 cell line (Figs. S2A and B in Supporting information). These results indicated that mPTT plus ACY-1215 can effectively reduce the survival of breast cancer cells. Then, cell apoptosis induced by mPTT with/without ACY-1215 were measured. The apoptosis of PdMo + Laser and PMH groups were 0.84% and 7.95%, respectively (Figs. 2C and D). Surprisingly, the early apoptosis rate of PMH + Laser group reached 29.64% (Figs. 2C and D). The results of fluorescence live/dead assay exhibited the same tendency (Fig. S2C in Supporting information). This suggested that mPTT plus ACY-1215 can significantly promote apoptosis. Furthermore, western blots were used to detect the marker proteins related to apoptosis. As shown in Figs. 2E–G, inhibition of HDAC6 by ACY-1215 induced a dose-dependent increasing of cleaved caspase-8 and PARP, and enhanced by mPTT effect, implying that the ACY-1215 combined with mPTT induced apoptosis through activating caspase-8 (a pro-apoptotic signal). Of note, HDAC6 plays an important role in maintaining cellular shape and polarity, intracellular transport, division, migration, and directional movement of the cells and angiogenesis [44,45]. For tumor therapy, the use of high doses of HDAC6 specific inhibitors will inevitably damage normal tissues. Meanwhile, the sole mPTT treatment also could not achieve efficient therapeutic effect for its mild character. Taken together, our data suggested that the combination of low-dose HDAC6 inhibitor ACY-1215 and mPTT effect is a potential option for cancer treatment.

  Figure 2

  

  Figure 2.  In vitro cytotoxicity analysis. (A, B) 4T1 cells were treated with increasing dose of PdMo or PMH (0, 5, 10, 20, 40 µg/mL). After 4 h, suffered to laser (0.5 W/cm², 15 min) and cell viability was measured by cell counting kit-8 (CCK8) at 24 h (A) and 36 h (B). Values represent means expressed as percentages compared with the untreated control. All data represent as the means ± standard deviation (SD) (n = 3). (C, D) 4T1 cells were treated with increasing dose of PdMo or PMH 20 µg/mL. After 4 h, suffered to laser and harvested 24 h late. Assessment of apoptosis by Annexin V and propidium iodine (PI). Quantification analysis of apoptosis (C) and Annexin V/PI assay on 4T1 cells (D). (E) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and harvested 24 h later. Cleaved PARP and caspase 8 were detected by Western blot. ac-Tubulin, acetyl-α-tubulin (Lys40); ac-H3, histone H3 total acetylation. (F) Quantification of cleaved PARP under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3, ^**P < 0.01). (G) Quantification of cleaved caspase 8 under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3, ^**P < 0.01).

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  HSPs play a critical role in mPTT-induced thermoresistance [17]. Once mild hyperthermia stimulated, heat-shock proteins, such as HSP90 and HSP70 could be up-regulated to promote misfolded and unfold proteins degradation through ubiquitin-proteasome system, which could prevent the accumulation of denatured proteins and maintain cellular homeostasis [24]. Thus we detected the protein level of HSP90 and HSP70 under experimental settings. As shown in Figs. 3A and B, both HSP70 and HSP90 were increased in mPTT treatment group. But in PMH + Laser group, this effect was reversed. Consistently, by immunostaining of HSP90, we also observed that the level of HSP90 was also significantly increased in PdMo + Laser treated cells and decreased in PMH + Laser treated cells (Fig. 3C). However, we did not obtain the same result in mRNA level (Fig. S3 in Supporting information). This difference may be caused by the rapid increase of the HSPs mRNA in the temperature shift [17,46]. In addition, post-translational modifications of HSP90, especially acetylation, might also act important role in this procedure [34]. Under physiological conditions, HSP90 interacts with the 'client proteins' and forms complexes to maintain their stability and function. HDAC6 knockdown or HDAC6 inhibition would promote the hyperacetylated HSP90 to dissociate with its client proteins, which result in the poly-ubquitination and degradation of HSP90 client protein through proteasome-dependent pathway [44,47]. Here, we detected the acetylation of HSP90 by immunoprecipitation. We found that the acetylation level of HSP90 was increased by ACY-1215 treatment, indicating the inhibition of HDAC6 impair the HSP deacetylation (Figs. 3D and E). At the same time, the hyperacetylated HSP90 facilitated the degradation of the client proteins, which in turn dysregulated the intracellular protein homeostasis and finally led to cell death.

  Figure 3

  

  Figure 3.  Mild-temperature photothermal therapy and HDAC6 inhibition in HSPs regulation and autophagy. (A) 4T1 cells were incubated with PdMo or PMH in 20 µg/mL. After 4 h, suffered to laser and harvested 24 h later. The protein level of HSP70 and HSP90 were detected by Western blot. (B) Quantification analysis of protein HSP70 and HSP90. All data represent as the means ± SD (n = 3, ^**P < 0.01). (C) 4T1 cells were incubated with PdMo or PMH in 20 µg/mL. After irradiation treated, cells were fixed and immunostained with anti-HSP90 antibodies. Scale bars, 0.45 µm. (DAPI: 4′, 6-diamidino-2-phenylindole). (D) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunoprecipitated with anti-HSP90. The acetylation of HSP90 was detected by Western blot. (Pan-ac: pan-acetylated-lysine). (E) Quantification analysis. All data represent as the means ± SD (n = 3, ^**P < 0.01). (F) 4T1 cells were treated with increasing dose of PdMo or PMH (10, 20 and 40 µg/mL). After 4 h, suffered to laser and harvested 24 h later. LC3B and P62 were detected by Western blot. (G) Quantification of LC3B and P62 under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3). (H) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunostained with anti-LC3B. Scale bars, 0.45 µm. (I) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunostained with anti-P62. Scale bars, 0.45 µm. (J) Possible mechanism of PMH plus mPTT-mediated cell death.

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  Autophagy is another critical process to remove misfolded and unfold proteins, large protein aggregates and damaged organelle lesion in eukaryotic cells [18]. While HDAC6 is a key regulator of autophagy to target damaged and misfolded proteins. Firstly, HDAC6 regulates aggresome formation, which induced by misfolded proteins [48]. Then, HDAC6 might favor the transport of lysosomes to the site of autophagy [48,49]. Pan-HDACi (SAHA) could induced autophagy by inhibiting the mammalian target of rapamycin (mTOR) complex 1, a key component in the regulation of autophagic process [50-52]. Here, we found that PMH also could decrease p-mTOR (Fig. 3F). Given that, we investigated the role of mPTT in HDAC6 inhibition-induced autophagy. We found that LC3BII (microtubule associated protein 1 light chain 3 beta type 2) was increased in the cells treated with PMH and PMH + Laser (Figs. 3F and G), respectively. These results suggested that both PMH and PMH plus mPTT could obviously induce autophagy. Interestingly, the level of P62 was decreased in PMH treatment group, but increased in the mPTT combination treatment group. Similarly, immunofluorescence data showed the similar results (Figs. 3H and I). These results indicated that ACY-1215 promoted autophagosome formation. However, when combined with mPTT, autophagosome fusion with the lysosome was inhibited, indicating the collapse of autophagy progress. Taken together, HDAC6 inhibition combined with mPTT induced cell death by inhibiting the proteasome-dependent degradation system and autophagy (Fig. 3J).

  Next, we established a mouse breast carcinoma model to further study the tumor accumulation of PMH in vivo. All animal experiments were carried out in strict accordance with the regulations of the Animal Ethical and Welfare Committee of Shenzhen University (AEWC-SZU). Based on the high-resolution of three-dimensional (3D) PA imaging, we assessed the PdMo-mediated mPTT and ACY-1215 efficiency in vivo. Firstly, we delineated PA signaling in tumor tissue. After intravenous injection (i.v.) of PMH, the PA signals were increased and reached the peak value (~7.8 fold) at about 4 h later and declined gradually (Figs. 4A and B). In addition, the PA images also showed that PMH could efficiently accumulate in the whole tumor region (Figs. 4C and D). These results suggested that the mPTT (1064 nm laser) could be performed under PA navigation.

  Figure 4

  

  Figure 4.  In vivo NIR-II PA tracking and in vivo combinatorial cancer therapy. (A) Time-dependent 3D-rendered images of tumor sites after intravenous injection (i.v.) of PMH. (B) Quantification of PA amplitude as a function of time, respectively. Data are presented as mean ± SD (n = 3, ^**P < 0.01). (C) 3D-rendered images of mice after treatment for 4 h and then 1064 nm laser irradiation for 10 min. (D) Quantification of PA values. (E) Schematic illustration of breast carcinoma model establishment and the therapeutic regimen. (F) In vivo photothermal images of mice after intravenous injection of control and PMH. (G) Temperature change curves of the laser-irradiated tumor tissues as a function of irradiation time; ^**P < 0.01 and ^***P < 0.001. (H) Relative tumor volume of the differentially-treated mice (n = 6, ^*P < 0.05 and ^**P < 0.01). (I) Representative images of tumor. (J) Relative tumor weight of the differentially-treated mice. (K) TUNEL, H&E and Ki-67 staining of tumor slices collected from different groups after various treatments indicated.

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  Due to the satisfactory therapeutic effect in vitro and the exciting PA imaging capability of PMH in vivo, the therapeutic effect of PMH in vivo was investigated (Fig. 4E). 4T1 tumor-bearing mice were randomly assigned into the following groups: 1) PBS; 2) Laser; 3) PdMo; 4) PdMo + Laser; 5) PMH; 6) PMH + Laser. After intravenous injection, the xenograft tumors (PdMo + Laser and PMH + Laser groups) were exposed in 1064 nm laser irradiation for 15 min (0.5 W/cm²) at 4 h post-injection according to the PA imaging results. Firstly, we explored the potential photothermal conversion in vivo. Laser-triggered tumor temperature dynamic change was real-time recorded by an infra-red (IR) thermal imaging camera. As shown in the thermal images and the time-temperature curves (Figs. 4F and G), upon 1064 nm laser irradiation, the tumor temperature increased rapidly and ultimately reached about 46 ℃, which fit in the range of mPTT. Then, the tumor size and body weight of the mice were recorded periodically. As shown in Fig. 4H, compared with other groups, the PMH + Laser treatment resulted in the restrained tumor growth (Fig. 4H), demonstrating that the combination of ACY-1215 and mPTT was more effective than treatment with either mPTT or ACY-1215 alone. In addition, 14 days later, tumors from all groups were harvested. The tumor images (Fig. 4I) and tumor weight (Fig. 4J) were consistent with above results. Of note, there was no obvious difference in body weight (Fig. S4A in Supporting information), indicating the relative biosafety of PMH.

  Subsequently, tumor tissues were performed histologically analyzed including terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL), hematoxylin eosin (H&E) and Ki-67 staining. As shown in Fig. 4K, in the combined treatment (PMH + Laser) group, the tumor tissues exhibited higher positive apoptotic/necrotic areas. Meanwhile, the H&E staining of heart, liver, spleen, lung and kidney showed no pathological changes (Fig. S4B in Supporting information). In addition, the blood biochemistry analysis of alanine aminotransferase (ALT), aspatate aminotransferase (AST), blood urea nitrogen (BUN) and creatinnine (CREA) were conducted to evaluate the biocompatibility. Our results showed that the PMH possessed fantastic biocompatibility (Figs. S4C–F in Supporting information). Taken together, ACY-1215, as a HDAC6 selective inhibitor, greatly enhanced anti-tumor efficacy of PTT at a mild condition.

  In summary, we have developed a photothermal theranostic nanomedicine (PMH), which integrated outstanding mPTT agents of PdMo, and the selective HDAC6 inhibitor (ACY-1215). PMH plus mPTT has synergistic effect on cancer treatment in vitro and in vivo. Mechanistically, PMH antagonizes mPPT-induced thermosresistance via restricting the elimination of misfolded and unfolded proteins, leading to cell death. Specifically, ACY-1215 impresses HSPs expression and reverses HDAC6-mediated deacetylation of HSP90, thereby impairing the misfolded and unfolded proteins degradation, which were regulated by HSPs. In general, when the removing of the accumulated harmful substances by proteasomal degradation is inhibited, autophagy is initiated. However, the combination of mPTT and ACY-1215 interferes autophagy process, leading to autophagy collapse. Taken together, the combination of ACY-1215 and mPTT will be a promising option for future clinical tumor therapy.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  Acknowledgments

  This work is financially supported by National Key R & D Program of China (Nos. 2020YFA0908800, 2018YFA0704000), Basic Research Program of Shenzhen (Nos. JCYJ20200109105620482, JCYJ20180507182413022), and Shenzhen Science and Technology Program (No. KQTD20190929172538530). We thank Instrumental Analysis Center of Shenzhen University (Lihu Campus).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.cclet.2023.108344.

  
  
• 1. [1]

     H.S. Jung, P. Verwilst, A. Sharma, et al., Chem. Soc. Rev. 47 (2018) 2280–2297. doi: 10.1039/c7cs00522a

  2. [2]

     V. Shanmugam, S. Selvakumar, C.S. Yeh, Chem. Soc. Rev. 43 (2014) 6254–6287.

  3. [3]

     H. Arami, S. Kananian, S.S. Gambhir, et al., Nat. Nanotechnol. 17 (2022) 1015–1022. doi: 10.1038/s41565-022-01189-y

  4. [4]

     X. He, Y. Hao, Z. Qian, et al., Nano Today 39 (2021) 101174.

  5. [5]

     K. Hu, L. Xie, M.R. Zhang, et al., Nat. Commun. 11 (2020) 2778.

  6. [6]

     X. Liu, H. Tao, Z. Liu, et al., Biomaterials 32 (2011) 144–151.

  7. [7]

     Y. Xiao, P. Sun, Z. Li, et al., Chin. Chem. Lett. 33 (2022) 5051–5055.

  8. [8]

     A. Lv, Q. Chen, H. Lin, et al., Chin. Chem. Lett. 32 (2021) 3653–3664.

  9. [9]

     Y. Liu, P. Bhattarai, Z. Dai, X. Chen, Chem. Soc. Rev. 48 (2019) 2053–2108.

  10. [10]

      H. Xiang, L. Zhao, Y. Zhao, et al., Nat. Commun. 12 (2021) 218.

  11. [11]

      X. Li, T. Yong, X. Yang, et al., Nat. Commun. 13 (2022) 2794.

  12. [12]

      Y. Jiang, J. Huang, C. Xu, K. Pu, Nat. Commun. 12 (2021) 742.

  13. [13]

      Z.H. Feng, Z.T. Li, X. Zhang, et al., Acta Biomater. 136 (2021) 495–507.

  14. [14]

      M. Chang, Z. Hou, J. Lin, et al., Angew. Chem. Int. Ed. 61 (2022) e202209245.

  15. [15]

      L. Huang, Y. Li, C. Sun, et al., Nat. Commun. 10 (2019) 4871.

  16. [16]

      B. Hildebrandt, P. Wust, H. Riess, et al., Crit. Rev. Oncol. Hematol. 43 (2002) 33–56.

  17. [17]

      K. Richter, M. Haslbeck, J. Buchner, Mol. Cell 40 (2010) 253–266.

  18. [18]

      K. Dokladny, O.B. Myers, P.L. Moseley, Autophagy 11 (2015) 200–213. doi: 10.1080/15548627.2015.1009776

  19. [19]

      S. Lu, T. Zhu, J. Hua, et al., Mol. Plant 14 (2021) 267–284.

  20. [20]

      T. Feng, L. Zhou, P. Huang, et al., Biomaterials 232 (2020) 119709.

  21. [21]

      N. Lu, P. Huang, X. Chen, et al., Biomaterials 126 (2017) 39–48.

  22. [22]

      Z. Deng, P. Huang, J. Lin, et al., Chin. Chem. Lett. 32 (2021) 2411–2414.

  23. [23]

      H.H. Kampinga, S. Bergink, Lancet Neurol. 15 (2016) 748–759.

  24. [24]

      H. Saibil, Nat. Rev. Mol. Cell Biol. 14 (2013) 630–642. doi: 10.1038/nrm3658

  25. [25]

      B. Tian, C. Wang, P. Yang, et al., Small 18 (2022) e2200786.

  26. [26]

      T. Sun, X. Cheng, Z. Xie, et al., Mater. Chem. Front. 3 (2019) 127–136. doi: 10.1039/c8qm00459e

  27. [27]

      R. Li, X. Hu, C. Dong, et al., Acta Biomater. 148 (2022) 218–229.

  28. [28]

      E. Schmitt, L. Maingret, C. Garrido, et al., Cancer Res. 66(2006) 4191–4197.

  29. [29]

      A. Aghdassi, P. Phillips, A. Saluja, et al., Cancer Res. 67 (2007) 616–625.

  30. [30]

      L. Whitesell, E.G. Mimnaugh, B. De Costa, C.E. Myers, L.M. Neckers, Proc. Natl. Acad. Sci. U. S. A. 91 (1994) 8324–8328. doi: 10.1073/pnas.91.18.8324

  31. [31]

      C. Hubbert, A. Guardiola, T.P. Yao, et al., Nature 417 (2002) 455–458.

  32. [32]

      T.Z. Zeleke, Q. Pan, C. Chiuzan, et al., Nat. Cancer 4 (2023) 257–275. doi: 10.1038/s43018-022-00489-5

  33. [33]

      J. Hey, M. Llamazares Prada, C. Plass, Nat. Cancer 4 (2023) 156–158. doi: 10.1038/s43018-022-00494-8

  34. [34]

      J.J. Kovacs, P.J. Murphy, T.P. Yao, et al., Mol. Cell 18 (2005) 601–607.

  35. [35]

      C. Dai, L. Whitesell, A.B. Rogers, S. Lindquist, Cell 130 (2007) 1005–1018.

  36. [36]

      Y. Ruan, L. Wang, Y. Lu, Drug Dev. Res. 82 (2021) 598–604. doi: 10.1002/ddr.21780

  37. [37]

      L. Santo, T. Hideshima, N. Raje, et al., Blood 119 (2012) 2579–2589. doi: 10.1182/blood-2011-10-387365

  38. [38]

      A.J. Yee, W.I. Bensinger, N.S. Raje, et al., Lancet Oncol. 17 (2016) 1569–1578.

  39. [39]

      J.E. Amengual, P. Johannet, O.A. O'Connor, et al., Clin. Cancer Res. 21 (2015) 4663–4675.

  40. [40]

      S. Lei, J. Zhang, P. Huang, et al., Nat. Commun. 13 (2022) 1298.

  41. [41]

      R. Long, K. Mao, Y. Xiong, et al., J. Am. Chem. Soc. 135 (2013) 3200–3207. doi: 10.1021/ja311739v

  42. [42]

      Z. Xi, X. Cheng, X. Xia, et al., Nano Lett. 20 (2020) 272–277. doi: 10.1021/acs.nanolett.9b03782

  43. [43]

      M. Luo, Z. Zhao, S. Guo, et al., Nature 574 (2019) 81–85. doi: 10.1038/s41586-019-1603-7

  44. [44]

      T. Li, C. Zhang, J. Yang, et al., J. Hematol. Oncol. 11 (2018) 111.

  45. [45]

      A. Valenzuela-Fernandez, J.R. Cabrero, J.M. Serrador, F. Sanchez-Madrid, Trends Cell Biol. 18 (2008) 291–297.

  46. [46]

      M.B. Eisen, P.T. Spellman, P.O. Brown, D. Botstein, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 14863–14868.

  47. [47]

      R. Rao, W. Fiskus, K. Bhalla, et al., Blood 112 (2008) 1886–1893. doi: 10.1182/blood-2008-03-143644

  48. [48]

      Y. Kawaguchi, J.J. Kovacs, T.P. Yao, et al., Cell 115 (2003) 727–738.

  49. [49]

      J.Y. Lee, H. Koga, T.P. Yao, et al., EMBO J. 29 (2010) 969–980. doi: 10.1038/emboj.2009.405

  50. [50]

      Y.L. Liu, P.M. Yang, C.C. Chen, et al., Autophagy 6 (2010) 1057–1065. doi: 10.4161/auto.6.8.13365

  51. [51]

      N. Gammoh, P.A. Marks, X. Jiang, Autophagy 8 (2012) 1521–1522. doi: 10.4161/auto.21151

  52. [52]

      N. Gammoh, D. Lam, C. Puente, et al., Proc. Natl. Acad. Sci. U. S. A. 109 (2012) 6561–6565. doi: 10.1073/pnas.1204429109

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  Scheme 1  Schematic illustration of the preparation of PdMo bimetallene with loading of ACY-1215 and its application in enhanced mild photothermal therapy.

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  Figure 1  Characterization of photophysical performance. (A) TEM image of PdMo bimetallene. (B) HRTEM image of PdMo bimetallene. (C) EDS elemental mapping of Pd and Mo in a single bimetallene nanosheet. (D) XRD spectra of PdMo bimetallene. High resolution XPS spectrum of I Pd 3d and Mo 3d for PdMo bimetallene. (F) UV–vis–NIR spectra of PdMo bimetalleneat different concentrations. (G) Concentration-dependent photothermal heating curves of PdMo bimetallene (0, 5, 10, 20, 40 µg/mL) under 1064 nm laser irradiation for 5 min. (H) Power density-dependent photothermal heating curves of PdMo bimetallene (0.2, 0.4, 0.6, 0.8, 1.0 W/cm²). (I) Photothermal stability of PdMo bimetallene (1064 nm, 0.6 W/cm²).

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  Figure 2  In vitro cytotoxicity analysis. (A, B) 4T1 cells were treated with increasing dose of PdMo or PMH (0, 5, 10, 20, 40 µg/mL). After 4 h, suffered to laser (0.5 W/cm², 15 min) and cell viability was measured by cell counting kit-8 (CCK8) at 24 h (A) and 36 h (B). Values represent means expressed as percentages compared with the untreated control. All data represent as the means ± standard deviation (SD) (n = 3). (C, D) 4T1 cells were treated with increasing dose of PdMo or PMH 20 µg/mL. After 4 h, suffered to laser and harvested 24 h late. Assessment of apoptosis by Annexin V and propidium iodine (PI). Quantification analysis of apoptosis (C) and Annexin V/PI assay on 4T1 cells (D). (E) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and harvested 24 h later. Cleaved PARP and caspase 8 were detected by Western blot. ac-Tubulin, acetyl-α-tubulin (Lys40); ac-H3, histone H3 total acetylation. (F) Quantification of cleaved PARP under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3, ^**P < 0.01). (G) Quantification of cleaved caspase 8 under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3, ^**P < 0.01).

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  Figure 3  Mild-temperature photothermal therapy and HDAC6 inhibition in HSPs regulation and autophagy. (A) 4T1 cells were incubated with PdMo or PMH in 20 µg/mL. After 4 h, suffered to laser and harvested 24 h later. The protein level of HSP70 and HSP90 were detected by Western blot. (B) Quantification analysis of protein HSP70 and HSP90. All data represent as the means ± SD (n = 3, ^**P < 0.01). (C) 4T1 cells were incubated with PdMo or PMH in 20 µg/mL. After irradiation treated, cells were fixed and immunostained with anti-HSP90 antibodies. Scale bars, 0.45 µm. (DAPI: 4′, 6-diamidino-2-phenylindole). (D) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunoprecipitated with anti-HSP90. The acetylation of HSP90 was detected by Western blot. (Pan-ac: pan-acetylated-lysine). (E) Quantification analysis. All data represent as the means ± SD (n = 3, ^**P < 0.01). (F) 4T1 cells were treated with increasing dose of PdMo or PMH (10, 20 and 40 µg/mL). After 4 h, suffered to laser and harvested 24 h later. LC3B and P62 were detected by Western blot. (G) Quantification of LC3B and P62 under 40 µg/mL treating condition. All data represent as the means ± SD (n = 3). (H) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunostained with anti-LC3B. Scale bars, 0.45 µm. (I) 4T1 cells were incubated with PdMo or PMH in 40 µg/mL. After 4 h, suffered to laser and immunostained with anti-P62. Scale bars, 0.45 µm. (J) Possible mechanism of PMH plus mPTT-mediated cell death.

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  Figure 4  In vivo NIR-II PA tracking and in vivo combinatorial cancer therapy. (A) Time-dependent 3D-rendered images of tumor sites after intravenous injection (i.v.) of PMH. (B) Quantification of PA amplitude as a function of time, respectively. Data are presented as mean ± SD (n = 3, ^**P < 0.01). (C) 3D-rendered images of mice after treatment for 4 h and then 1064 nm laser irradiation for 10 min. (D) Quantification of PA values. (E) Schematic illustration of breast carcinoma model establishment and the therapeutic regimen. (F) In vivo photothermal images of mice after intravenous injection of control and PMH. (G) Temperature change curves of the laser-irradiated tumor tissues as a function of irradiation time; ^**P < 0.01 and ^***P < 0.001. (H) Relative tumor volume of the differentially-treated mice (n = 6, ^*P < 0.05 and ^**P < 0.01). (I) Representative images of tumor. (J) Relative tumor weight of the differentially-treated mice. (K) TUNEL, H&E and Ki-67 staining of tumor slices collected from different groups after various treatments indicated.

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