Ru(II)-modified TiO₂ nanoparticles for hypoxia-adaptive photo-immunotherapy of oral squamous cell carcinoma.

{"full_text": " Biomaterials 289 (2022) 121757\n\n\n Contents lists available at ScienceDirect\n\n\n Biomaterials\n journal homepage: www.elsevier.com/locate/biomaterials\n\n\n\n\nRu(II)-modified TiO2 nanoparticles for hypoxia-adaptive\nphoto-immunotherapy of oral squamous cell carcinoma\nJia-Ying Zhou a, b, 1, Wen-Jin Wang c, 1, Chen-Yu Zhang a, b, 1, Yu-Yi Ling c, 1, Xiao-Jing Hong a, b,\nQiao Su d, Wu-Guo Li d, Zong-Wan Mao c, ****, Bin Cheng a, b, ***, Cai-Ping Tan c, **, Tong Wu a, b, *\na\n Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, 510055, PR China\nb\n Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, 510080, PR China\nc\n MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of Chemistry, Sun Yat-sen University, Guangzhou, 510006, PR China\nd\n Animal Experiment Center, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, 510080, PR China\n\n\n\n\nA R T I C L E I N F O A B S T R A C T\n\nKeywords: The alternations in the hypoxic and immune microenvironment are closely related to the therapeutic effect and\nTitanium dioxide prognosis of oral squamous cell carcinoma (OSCC). Herein, a new nanocomposite, TiO2@Ru@siRNA is con\u00ad\nRuthenium structed from a ruthenium-based photosensitizer (Ru) modified-TiO2 nanoparticles (NPs) loaded with siRNA of\nPhoto-immunotherapy\n hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1). Under visible light irradiation, TiO2@Ru@siRNA can elicit both Type I\nOral squamous cell carcinoma\nHypoxia adaptation\n and Type II photodynamic effects, which causes lysosomal damage, HIF-1\u03b1 gene silencing, and OSCC cell\n elimination efficiently. As a consequence of hypoxia relief and pyroptosis induction, TiO2@Ru@siRNA reshapes\n the immune microenvironment by downregulation of key immunosuppressive factors, upregulation of immune\n cytokines, and activation of CD4+ and CD8+ T lymphocytes. Furthermore, patient-derived xenograft (PDX) and\n rat oral experimental carcinogenesis models prove that TiO2@Ru@siRNA-mediated photodynamic therapy\n significantly inhibits the tumor growth and progression, and markedly enhances cancer immunity. In all, this\n study presents an effective hypoxia-adaptive photo-immunotherapeutic nanosystem with great potential for\n OSCC prevention and treatment.\n\n\n\n\n1. Introduction primary cancer prevention and treatment modality for OSCC patients\n with early stages of the disease [5,6].\n Surgical resection remains the primary therapy at present for oral Hypoxia is a common phenomenon in solid tumors, and OSCC is\nsquamous cell carcinoma (OSCC), with chemotherapy and radiotherapy characterized by large areas of tumoral necrosis and local hypoxia,\nas the main adjuvant treatments [1]. However, the 5-year overall sur\u00ad which causes low response to chemotherapy or even drug resistance [7].\nvival rate still remains 60% with such multimodality treatments, which Immunosuppression is another factor accounting for the low-response\ncause significant mutilation leading to life quality reduction simulta\u00ad therapy and poor prognosis of OSCC [8]. Bioinformatics analysis\nneously [2]. Photodynamic therapy (PDT), an effective therapeutic shows that the expression of hypoxia-inducible factor-1\u03b1 (HIF-1\u03b1) and\nmodality in spatiotemporal selectivity superficial tumor, which is based programmed death ligand-1 (PD-L1) is significantly more abundant in\non photosensitizers (PSs) constructed with nanomaterials, has become OSCC patients with poor prognosis (p < 0.05) in public database\npopular for OSCC prevention and treatment [3,4]. Because of its ad\u00ad (Figure S1). Moreover, HIF-1\u03b1 can upregulate the expression of PD-L1 in\nvantages such as higher specificity and repeatability, lower drug resis\u00ad glioma, endometrial carcinoma and lung cancer [9\u201311]. Thus hypoxia\ntance and fewer side effects, PDT has become a promising technique as a adaption combined with PD-L1 blockage may be one of the most\n\n\n * Corresponding authors. Hospital of Stomatology, Guanghua School of Stomatology, Sun Yat-sen University, Guangzhou, 510055, PR China.\n ** Corresponding author.\n *** Corresponding author.\n **** Corresponding author.\n E-mail addresses: cesmzw@mail.sysu.edu.cn (Z.-W. Mao), chengbin@mail.sysu.edu.cn (B. Cheng), tancaip@mail.sysu.edu.cn (C.-P. Tan), wutong23@mail.sysu.\nedu.cn (T. Wu).\n 1\n These authors contributed equally to this work.\n\nhttps://doi.org/10.1016/j.biomaterials.2022.121757\nReceived 15 June 2022; Received in revised form 2 August 2022; Accepted 18 August 2022\nAvailable online 24 August 2022\n0142-9612/\u00a9 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-\nnc-nd/4.0/).\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nScheme 1. (A) Construction of the nanocomposite TiO2@Ru@siRNA. (B) Action mechanism of TiO2@Ru@siRNA. ROS: Reactive oxygen species; HIF-1\u03b1: Hypoxia-\ninducible factor-1\u03b1; HMGB1: High mobility group box-1 protein; NF-\u03baB: Nuclear factor-kappa B; PD-L1: Programmed death ligand-1; IL-24: Interleukin-24; GSDMD:\nGasdermin D; PDT: Photodynamic therapy; IFN-\u03b3: Interferon-gamma.\n\n\npromising approaches to activate antitumor immunity. other potential mechanisms [25,28].\n Since most of PDT treatments are based on the production of singlet Titanium dioxide (TiO2) is one of the most widely used nano\u00ad\noxygen (1O2), one of the biggest challenges of PDT is modulating the materials in human life, and TiO2-based materials have also been widely\nhypoxic microenvironment [12\u201314]. Several strategies have been investigated as PSs in PDT since the discovery of its photocatalytic ac\u00ad\ndeveloped to combat tumor hypoxia, including transporting or genera\u00ad tivity in 1972 [30\u201332]. Under radiation, TiO2 can usually damage cells\ntion of oxygen at tumor sites, such as hyperbaric oxygen, oxygen by reacting with water to produce reactive oxygen species (ROS). Due to\ntransportation by nanocapsules or perfluorocarbon, dissociation of ox\u00ad its excellent biocompatibility, TiO2 is also used as a drug delivery system\nygen from oxyhemoglobin, H2O2 degradation, water decomposition and for prosthesis implantation and early diseases treatment including can\u00ad\nreduction of oxygen consumption [15\u201319]. Developing PSs through cer [31,32]. Unfortunately, the light response range of TiO2 is limited to\nO2-independent mechanism is another feasible strategy to overcome the ultraviolet region with poor permeability [33]. Different modifica\u00ad\ntumor hypoxia [20\u201324]. Especially, an oxygen independent PS TOO\u00ad tions are used to extend the absorption wavelength of TiO2 even to the\nKAD\u00ae-Soluble (a derivative of the photosynthetic pigment bacterio\u00ad near-infrared range [34\u201337]. The transfer of photo-electrons from the\nchlorophyll \u03b1 with palladium as metal center), which mainly produces modifiers (antennas or nanocomposites) to the conduction band (CB) of\nsuperoxide anion (\u2022O\u22122 ) or hydroxyl radical (\u2022OH), has been clinically TiO2 can achieve efficient electron hole separation, which is conducive\napproved in several countries [25\u201327]. to the reaction between electrons and O2 to produce 1O2, while the re\u00ad\n Metal polypyridyl complexes have attracted more and more atten\u00ad action between holes and water molecules can produce \u2022OH and a va\u00ad\ntion as potent PSs, among which a Ru(II) complex TLD-1433, has riety of reactive oxygen radicals [34\u201337]. These properties make\nentered phase II clinical trial for non-muscle-invasive bladder cancer in modified TiO2 nanoparticles (NPs) ideal biocompatible PSs that can\nCanada [28,29]. The construction of TLD-1433 utilizes the high effi\u00ad exert PDT effects in both O2-dependent and -independent manners.\nciency of the intersystem crossing process of the Ru(II) center to obtain a Herein, we constructed a hypoxia-adaptive nanocomposite\npopulated long-lived excited triplet intraligand charge transfer state, TiO2@Ru@siRNA for prevention and treatment of OSCC. TiO2@R\u00ad\nwhich can generate 1O2 in high quantum yields or interact with the u@siRNA is constructed by coupling a Ru complex with a similar\nbiological environments by electron transfer reactions [28,29]. Inter\u00ad structure as TLD1433 to TiO2 NPs, and a siRNA targeting HIF-1\u03b1 is\nestingly, TLD-1433 has phototoxic effects under hypoxia, and it can further loaded (Scheme 1). TiO2@Ru@siRNA can produce ROS through\ncause DNA damage in the absence of oxygen, indicating that it may have both type I and type II PDT under visible light (525 nm) irradiation. With\n\n 2\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\na photocytotoxicity index (PI) up to more than 2000, TiO2@R\u00ad Cell imaging experiments were carried out on a confocal microscope\nu@siRNA-mediated PDT causes lysosomal damage to effectively pro\u00ad (Zeiss LSM-710, ZEISS, Germany). Cell viability assay was determined\nmote siRNA escape and induce pyroptosis of OSCC cells, which activates using a microplate spectrophotometer (Infinite M200 Pro, Tecan,\nmultifaceted cancer immune responses. In particular, the PDT effects of Switzerland). Flow cytometry was performed on a LSRFortessa (BD\nTiO2@Ru@siRNA are confirmed in patient-derived xenograft (PDX) Biosciences, USA) and Cytoflex flow cytometer (Becton Dickinson, USA).\nmodel and 4-nitroquinoline-1-oxide (4NQO) induced rat oral carcino\u00ad\ngenesis model. Collectively, TiO2@Ru@siRNA has outstanding PDT 2.3. Synthesis\nperformance and immunomodulatory function for OSCC through hyp\u00ad\noxia adaptation and cancer immune microenvironment optimization. cis-[RuCl2(DMSO)4], and TTIP (2-([2,2\u2019:5\u2032 ,2\u2032\u2032 -terthiophen]-5-yl)-1H-\nThe possibility of potential clinical transformation of this system is still imidazo[4,5-f][1,10]phenanthroline) were synthesized by literature\nunder exploration. methods [38,39].\n [Ru(dimbpc)2Cl2]: dimbpc (1.36 g, 5.00 mmol) and cis-\n2. Materials and methods [RuCl2(DMSO)4] (1.21 g, 2.50 mmol) were dispersed in N,N-\n dimethylformamide (DMF; 20 mL). Then, the reaction mixture was\n2.1. Materials refluxed for 6 h. Then, after evaporation of DMF, 50 mL pre-cold acetone\n was added. The solution was stored at 0 \u25e6 C for 2 h. And the solid was\n All reagents were commercially available and used without further filtered and washed with acetone and water until the filtrate became\npurification. RuCl3\u20223H2O was obtained from Adams Chemical, China. colorless. [Ru(dimbpc)2Cl2] was obtained as a black solid. Yield: 1.17 g\nDimethyl [2,2\u2032 -bipyridine]-4,4\u2032 -dicarboxylate (dimbpc) and amino\u00ad (63.9%).\npropyltriethoxysilane (APTEs) were purchased from Bidepharm (China). [Ru(dimbpc)2(TTIP)](Cl)2 (Rua): [Ru(dimbpc)2Cl2] (723.53 mg,\n5-formyl-2,2\u2019:5\u2032 ,2\u2032\u2032 -terthiophene and 1,10-Phenanthroline-5,6-dione 1.00 mmol) and TTIP (446.60 mg, 1.00 mmol) were dispersed in 4 mL\nwere bought from Energy Chemical (China). Methyl Thiazol Tetrazo\u00ad MeOH. The reaction was rapidly heated to 150 \u25e6 C in a microwave\nlium (MTT) was purchased from J&K Chemical (China). Reactive Oxy\u00ad reactor and maintained for 10 min. Then, the reaction was filtered. The\ngen Species Assay Kit (2\u2032 ,7\u2032 -dichlorodihydrofluorescein diacetate, liquid phase was collected and purified through flash chromatography\nDCFH-DA), 4% Paraformaldehyde Fix Solution (4% PFA fix solution), on a silica column (MeCN/H2O/20%KCl, v/v/v = 90/10/1). Evapora\u00ad\nRIPA-Lysis Buffer, SDS-PAGE Sample Loading Buffer, hematoxylin and tion of the solvent under nitrogen afforded the product as a red solid.\neosin were obtained from Beyotime Biotechnology (China). RNeasy Yield: 479.4 mg (43.1%). 1H NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm) 14.60\nMinikit was bought from Qiagen (Germany). PrimeScript RT Master Mix (s, 1H), 9.41 (d, J = 7.2 Hz, 4H), 9.04 (d, J = 6.1 Hz, 2H), 8.13\u20138.06 (m,\nwas bought from TaKaRa (China). SYBR Green Master Mix was bought 4H), 7.96 (d, J = 3.6 Hz, 2H),7.90 (dd, J = 5.6 Hz and 1.2 Hz, 2H), 7.85\nfrom Yeasen (China). 2-Phenyl-1,2-benzisoselenazol-3(2H)-one (Ebse\u00ad (d, J = 6.0 Hz, 2H), 7.75 (d, J = 7.2 Hz, 1H), 7.58 (d, J = 12.0 Hz, 1H),\nlen) was purchased from MedChemExpress (China). 4,5-Dihydroxy-1,3- 7.54 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 2.8 Hz, 1H) 7.40 (dd, J = 3.8, 0.7\nbenzenedisulfonic acid disodium salt monohydrate (Tiron), Sodium Hz, 1H), 7.35 (d, J = 2.8 Hz, 1H), 7.14 (dd, J = 3.8 and 2.8 Hz, 1H), 4.01\npyruvate (NaP), D-mannitol, phosphate buffered saline (PBS), dimethyl (s, 6H), 3.94 (s, 6H). 13C NMR (400 MHz, DMSO\u2011d6) \u03b4 (ppm) 163.90,\nsulfoxide (DMSO), phorbol 12-myristate 13-acetate (PMA), ionomycin 163.81, 157.27, 157.00, 152.95, 152.56, 147.60, 138.63, 138.11,\nand 4-nitroquinoline-1-oxide (4NQO) were purchased from Sigma- 137.99, 136.48, 135.60, 134.12, 130.77, 128.46, 126.64, 126.17,\nAldrich (USA). Fetal bovine serum (FBS), trypsin, Dulbecco\u2019s modified 126.08, 125.12, 124.61, 123.87, 53.29, 53.23. ESI-MS m/z (CH3OH):\nEagle\u2019s medium (DMEM), Roswell Park Memorial Institute Medium calculated for [M\u2212 2(Cl)]2+ (C53H38N8O8RuS2+ 3 ) 556.05, found 556.44;\n(RPMI1640 Medium), streptomycin, and LysoTracker Deep Red (LTDR) calculated for [M\u2212 2(Cl)\u2013H]+ (C53H37N8O8RuS2+ 3 ) 1111.09, found\nwere purchased from Gibco (Thermo Fisher Scientific, USA). FITC anti- 1111.46.\nhuman CD4, Brilliant Violet 605 anti-human CD8, BV421 anti-human [Ru(bpc)2(TTIP)](Cl2) (Ru) (bpc = [2,2\u2032 -bipyridine]-4,4\u2032 -dicar\u00ad\nIFN-\u03b3, Brefeldin A (BFA), Human TruStain FcX (Fc Receptor Blocking boxylic acid): [Ru(dimbpc)2(TTIP)] (Cl)2 (118.3 mg, 0.1 mmol) was\nSolution), Fixation Buffer, Intracellular Staining Permeabilization Wash dispersed in a 1 N NaOH solution, and refluxed for 1 h. After then, the\nBuffer, and Permeabilization Wash Buffer were purchased from Bio\u00ad reaction was cooled to 0 \u25e6 C and acidized with 1 N HCl to cat. pH 3 was to\nLegend (USA). Alexa Fluor\u00ae 488 Rabbit monoclonal to HMGB1, Alexa obtain the precipitation of Ru. The precipitation was filtered and\nFluor\u00ae 647 Rabbit monoclonal to Calreticulin (CRT), anti\u2013HIF\u20131 alpha washed with 10 mL MeOH. Then, the solid was dissolved using 1 N\nantibody, anti-GSDMD antibody, anti-Cleaved Caspase-1 antibody, anti- NaOH to remove insoluble composition. The liquid phase was acidized\n\u03b2-Actin antibody, anti\u2013NF\u2013\u03baB p65 antibody, anti-PD-L1 antibody, anti- and afforded the precipitation of Ru. The precipitation was filtered and\nKi67 antibody, and anti-CD3 antibody were purchased from Abcam washed with MeOH and water, and then dried in vacuo to obtain the Ru\n(UK) and used as recommended by the manufacturer. as a dark red solid. Yield: 107.9 mg (91.2%). 1H NMR (400 MHz, MeOD)\n \u03b4 9.13 (dd, J = 8.3, 1.0 Hz, 2H), 9.11\u20139.03 (m, 4H), 8.00 (d, J = 5.8 Hz,\n2.2. Instruments 2H), 7.91\u20137.85 (m, 4H), 7.81 (d, J = 3.8 Hz, 1H), 7.74\u20137.63 (m, 6H),\n 7.33 (dd, J = 5.1, 0.9 Hz, 1H), 7.30\u20137.20 (m, 3H), 7.16 (d, J = 3.8 Hz,\n ESI-MS was carried out on a Thermo Scientific LTQ linear ion trap 1H), 7.05 (dd, J = 5.1, 3.7 Hz, 1H). 13C NMR (101 MHz, MeOD) \u03b4\nmass spectrometer. 1H NMR and 13C NMR were recorded by a Bruker 170.19, 159.01, 158.82, 158.72, 152.87, 152.49, 148.71, 148.59,\nAdvance III 400 MHz spectrometer (Germany). Chemical shifts were 148.41, 145.56, 140.07, 139.79, 138.23, 137.78, 137.60, 137.35,\nreferenced relative to the internal solvent signals. The UV\u2013Vis absorp\u00ad 131.75, 129.07, 127.91, 127.74, 127.43, 126.66, 126.07, 125.73,\ntion spectra were obtained on a Varian Cary 100 spectrophotometer 125.54, 125.28, 124.83, 124.66, 124.61. ESI-MS m/z (CH3OH): calcu\u00ad\n(Agilent technologies, USA). Microanalyses (C, H, and N) were carried lated for [M\u2212 2(Cl)]2+ (C49H30N8O8RuS2+ 3 ) 528.02, found 528.35;\nout using an Elemental Vario EL CHNS analyzer (Germany). The fluo\u00ad calculated for [M\u2212 2(Cl)\u2013H]+ (C49H29N8O8RuS2+ 3 ) 1055.03, found\nrescence emission spectra were obtained on an FLS 980 combined 1055.13.\nfluorescence lifetime and steady state spectrometer (Edinburgh Instru\u00ad TiO2-APTEs: Using reported amino silanization method [40], the\nment, UK). The Zeta potentials and hydrodynamic diameters of TiO2- TiO2 nanoparticles were modified with an activated silane coupling\nAPTEs, TiO2@Ru and TiO2@Ru@siRNA samples dissolved in deion\u00ad agent APTEs to obtain positively charged TiO2-APTEs. In brief, the TiO2\nized water were measured by EliteSizer (Brookhaven Instruments; USA) suspension was prepared by adding 2.1 mg TiO2 nanoparticles to 7 mL\nat 25 \u25e6 C. Morphology of the nanoparticles was analyzed using a Trans\u00ad of methanol solution, followed by an ultrasonic dispersion for 15 min. A\nmission Electron Microscopy (TEM, T12, FEI Tecnai G2 Spirit, Holland). reaction liquid was prepared by adding 1 mL APTEs to 14 mL methanol\n\n 3\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 1. Characterization of the TiO2@Ru NPs. (A) TEM image of the TiO2@Ru NPs. Scale bar: 100 nm. (B) IR absorption spectra of TiO2, TiO2-APTEs and TiO2@Ru.\n(C) Hydrodynamic diameter of the TiO2@Ru nanoparticles and their distribution measured by DLS. (D) Zeta potentials of the TiO2, TiO2-APTEs and TiO2@Ru NPs\nwith different loading ratios. (E) Quantitative EDX analysis of TiO2@Ru. (F) The photocatalytic water splitting mechanism on TiO2@Ru under visible light irra\u00ad\ndiation (525 nm, 15 mW cm\u2212 2, 30 min). The decay curves of MB absorption at 660 nm TiO2@Ru: 2 \u03bcg/mL; MB: 100 \u03bcM. (G) The ESR spectra of \u2022OH generated by\nTiO2@Ru, Ru and TiO2 after light irradiation (525 nm, 15 mW cm\u2212 2, 30 min) using DMPO as the \u2022OH trap. TiO2@Ru: 2 \u03bcg/mL; DMPO: 100 \u03bcM. (H) The\ndegradation rate of ABDA photosensitized by TiO2@Ru, Ru and [Ru(bpy)3]Cl2 in aerated PBS as shown by the decrease in the absorption maxima of ABDA at 380 nm\nTiO2@Ru: 2 \u03bcg/mL; ABDA: 100 \u03bcM. (I) The mechanisms of TiO2@Ru NPs to generate ROS through both Type I and Type II pathways. (J) Agarose gel electrophoresis\nof free siRNA (2 \u03bcM) and TiO2@Ru complex (200 \u03bcg/mL) mixed at different pH. (K) Zeta potentials of the TiO2@Ru before and after loading of HIF-1\u03b1 siRNA.\n\n\nsolution, after which 0.7 mL ammonia solution was also added to the 10 mL DMF and water three times, respectively. Then the nanoparticles\ncombination that had been stirred for 15 min. Then the TiO2 suspension were dried in vacuo overnight.\nwas added in drops into the reaction liquid with vigorous stirring, and TiO2@Ru@siRNA: The TiO2@Ru-APTEs nanoparticles were\nthe mixture was then stirred for 24 h at room temperature to form dispersed in pH 6.5 PBS buffer to a final concentration of 200 \u03bcg/mL.\nTiO2-APTEs. The HIF-1\u03b1-siRNA was added to the reaction until final concentration\n TiO2@Ru: Ru (16.9 mg, 0.015 mmol) and 2-(7-Azabenzotriazol-1- was 2 \u03bcM. The mixture was sired at 4 \u25e6 C for 30 min. After then, the\nyl)-N,N,N\u2032 ,N\u2032 -tetramethyluronium hexafluorophosphate (11.4 mg, 0.03 nanoparticles were centrifuged at 10,000 g for 10 min and washed with\nmmol) were dissolved in DMF (2 mL), followed by addition of N,N- pH 6.5 PBS buffer three times. At last, the nanoparticles were dispersed\nDiisopropylethylamine (10.8 \u03bcL, 0.06 mmol). A solution of well with DEPC water.\ndispersive TiO2-APTEs (67 mg) in DMF (10 mL) was added and the See supplementary information for characterization details.\nreaction mixture was stirred overnight. Then, the reaction mixture was\ncentrifuged at 10,000 g for 10 min. The centrifugate was washed with\n\n\n 4\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n2.4. PDX model The p25 TiO2 nanoparticles were embellished by APTEs, which\n endowed TiO2 with a positive potential (Scheme S2). Ru was coupled\n The PDX model was established as described previously [41]. Tumor onto TiO2-APTEs NPs to obtain TiO2@Ru through amidation, and the\nsamples were collected at the Department of Head and Neck Surgery, maximum loading capacity was calculated to be 0.315 mg/mg (Ru/\nSun Yat-sen University Cancer Center. Prior informed consent was ob\u00ad TiO2-APTEs; Figure S5). TEM image of TiO2@Ru shows a well-defined\ntained from OSCC patient and the research was approved by the Medical cube shape with a diameter of about 40 nm (Fig. 1A).\nEthics of Committee of Hospital of Stomatology, Sun Yat-sen university After conjugation of Ru, the vibration peaks of 3004 nm (\u03bdC-H), 1655\n(KQEC-2021-64-2). In brief, freshly resected tumors were intensively nm (\u03bdC\u2013 \u2013 O), 1437 nm (\u03b2C-H) and 1314 nm (\u03bdC-N) were detected in the\nwashed and cut into small pieces (diameter, 0.8\u20131.5 mm) in antibiotic infrared (IR) spectrum of TiO2@Ru, indicating that Ru has been suc\u00ad\ncontaining DMEM. Then, the tumor pieces were implanted subcutane\u00ad cessfully loaded on TiO2 (Fig. 1B). The UV\u2013Vis spectrum of TiO2@Ru\nously into the flanks of nude mice (P0 xenografts). The tumor size was shows Ru modification enhances the visible light absorption of the TiO2\nmeasured using a vernier caliper when the inoculated tissue grew into NPs (Figure S6). The diameter measured by dynamic light scattering\nthe tumor. The tumor volume (mm3) was calculated by the following (DLS, Fig. 1C) is about 82 nm, which is larger than that measured by\nformula: V = a \u00d7 b2/2, where V represented the tumor volume, and a TEM, as DLS gives a hydrodynamic size that corresponds to the core and\nand b were the longest and shortest tumor diameters, respectively. When the swollen corona of the micelles, while TEM often gives a size of the\nthe tumor size reached 1500 mm3, the tumors were dissected, processed core for micelles in a dried state.\nand reinjected for expansion (P1 xenografts). This process was further The zeta potentials of p25 TiO2 and TiO2-APTEs are \u2212 7.41 \u00b1 1.22\nrepeated, and the animal study was performed with P6 xenografts. When and +27.65 \u00b1 2.46 mV, respectively (Fig. 1D). The zeta potential is\nthe xenografts reached a mean size of 200 mm3, the mice were ran\u00ad decreased with the increase in the Ru loading. In order to preserve the\ndomized into 6 groups (5 mice per group) and treated as follows: (A) positive surface charges of the TiO2@Ru NPs for optimized loading of\ncontrol (50 \u03bcL saline); (B) control (50 \u03bcL saline) + light; (C) TiO2@Ru negatively charged siRNA, 80% of the maximum loading capacity was\n(50 \u03bcL, 20 mg/kg); (D) TiO2@Ru (50 \u03bcL, 20 mg/kg) + light; (E) used as the optimum loading ratio (0.252 mg/mg; Ru/TiO2-APTEs).\nTiO2@Ru@siRNA (50 \u03bcL, 20 mg/kg); (F) TiO2@Ru@siRNA (50 \u03bcL, 20 Quantitative energy dispersive X-ray (EDX) analysis shows the presence\nmg/kg) + light. Mice were treated with Ru complexes twice at day 0 and of Ti, Ru, C, N, O and S elements in TiO2@Ru, and the mass proportion\nday 7, and PDT was conducted with a 525 nm laser (15 mW cm\u2212 2, 60 of Ru element is 1.6% (Fig. 1E). The weight percentage of the Ru coating\nmin) after intratumorally injection. Body weight and the subcutaneous measured by energy dispersive X-ray spectrometer (EDS) is 18.0%,\ntumor size were measured every other day. The mice were sacrificed 23 which is similar to the weight percentage (25.2%) calculated by loading\ndays after treatment. Tumor tissues were removed, weighed, fixed in capacity measured by UV absorption calculation (Figure S5).\n10% buffered formalin, and embedded in paraffin. And major organs TiO2 NPs can degrade the H2O producing \u2022OH, we next studied the\nincluding the heart, liver, spleen, lung, and kidney were removed and water splitting capability of TiO2@Ru under visible light irradiation\nembedded in paraffin for histopathological assessment. (525 nm, 15 mW cm\u2212 2, 30 min) using methylene blue as the indicator\n (Fig. 1F). Besides, the generation of \u2022OH upon visible light irradiation\n2.5. 4NQO-induced rat oral carcinogenesis model (525 nm, 15 mW cm\u2212 2, 30 min) is also been confirmed by electron spin\n resonance (ESR) using 5,5-dimethylpyrroline N-oxide (DMPO) as the\n The 4NQO-induced rat oral carcinogenesis model was established as trap for \u2022OH (Fig. 1G). Both results indicate that TiO2@Ru can effec\u00ad\ndescribed previously [42]. Male Sprague-Dawley (SD) rats (male, 4 tively generate \u2022OH after irradiation with visible light. The significant\nweeks) were fed daily with 20 ppm 4NQO solution in their drinking decrease of the absorbance of 9,10-anthracenediyl-bis (methylene)\nwater from week 0 to week 16. After the 16-week carcinogen treatment, dimalonic acid (ABDA, an 1O2 indicator) in the presence of TiO2@Ru\nthe drinking water was switched to distilled water. The rats were divided after irradiation shows that TiO2@Ru can also efficiently photosensitize\ninto 4 groups (5 rats per group) and treated as follow: (A) control (150 O2 to generate 1O2. The 1O2 yield for TiO2@Ru is 0.38, which is higher\n\u03bcL saline); (B) control (150 \u03bcL saline) + light; (C) TiO2@Ru@siRNA than the 1O2 yield of Ru (0.28) under the same conditions (Fig. 1H).\n(150 \u03bcL, 20 mg/kg); (D) TiO2@Ru@siRNA (150 \u03bcL, 20 mg/kg) + light. The phosphorescent lifetime of Ru is greatly shortened by loading\nRats were treated with Ru complexes twice at the start of week 17 and onto TiO2 NPs, which indicates there exists energy transfer between Ru\nweek 18, and PDT was conducted with a 525 nm laser (15 mW cm\u2212 2, 60 and TiO2 NPs (Figure S7). Therefore, we propose that due to the energy\nmin) after submucosal injection. At week 21, the rats were sacrificed and transfer between the antenna (Ru) and TiO2, TiO2 can be excited by\nthe tongues were dissected, and a longitudinal mid-lingual incision was visible light. Ru transfers the energy to the CB of TiO2 for effective\nmade. Half of the specimens were fixed in 10% buffered formalin, electron hole separation (Fig. 1I). On the one hand, the process facilities\nembedded in paraffin and cut into 4 mm sections for hematoxylin and the energy transfer from TiO2 to O2 for the generation of 1O2. On the\neosin (H&E) staining to confirm the pathological diagnosis. The other other hand, \u2022OH can be produced from the reaction between TiO2 holes\nhalf of the specimens were stored at \u2212 80 \u25e6 C. and H2O. Moreover, the conjugated Ru molecules can also produce \u2022OH\n All of the animal procedures were conducted in accordance with the and 1O2 upon irradiation (Fig. 1G and H).\nGuidelines for the Care and Use of Laboratory Animals and were The siRNA of HIF-1\u03b1 was loaded onto TiO2@Ru through electro\u00ad\napproved by the Institutional Animal Care and Use Committee at Sun static interaction to afford TiO2@Ru@siRNA NPs. Agarose gel elec\u00ad\nYat-sen University. trophoresis shows that 2.0 \u03bcM siRNA can be successfully loaded onto\n See supplementary information for the other experimental TiO2@Ru NPs (200 \u03bcg/mL) at pH 6.5 (Fig. 1J), and the Zeta potential of\nmethods. TiO2@Ru@siRNA is +1.14 \u00b1 0.37 mV (Fig. 1K). TEM image of\n TiO2@Ru@siRNA shows a well-defined cube shape with a diameter of\n3. Results and discussion about 50 nm (Figure S8A), and the hydrodynamic diameter for\n TiO2@Ru@siRNA is about 100 nm as measured by DLS (Figure S8B).\n3.1. Synthesis and characterization of TiO2@Ru@siRNA After being placed in FBS for 7 days, no precipitate is observed and\n no significant change in the size of TiO2@Ru@siRNA can be detected,\n The Ru complex (Ru) was synthesized by the microwave reaction of which proves that it has good biological stability (Figure S9).\nthe precursor [Ru(dimbpc)2]Cl2 with ligand TTIP (Scheme S1). The\ncrude product (Rua) was hydrolyzed in NaOH to obtain the target\nproduct Ru. Ru was characterized by ESI-MS, 1H NMR and 13C NMR\n(Figure S2\u2013S4).\n\n 5\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\nTable 1\nCytotoxicity (IC50, \u03bcg/mL)[a] of the NPs on different cell lines[b].\n Conditions HN6 HSC-6 DOK\n\n\n Dark Light PI[c] Dark Light PI[c] Dark Light PI[c]\n\n Normoxia (20% O2)\n Ru \uff1e112 0.26 \u00b1 0.01 \uff1e431 \uff1e112 0.29 \u00b1 0.01 \uff1e386 \uff1e112 0.25 \u00b1 0.01 \uff1e448\n TiO2@Ru \uff1e500 0.31 \u00b1 0.02 \uff1e1613 \uff1e500 0.64 \u00b1 0.03 \uff1e781 \uff1e500 0.71 \u00b1 0.06 \uff1e704\n TiO2@Ru@siRNA \uff1e500 0.18 \u00b1 0.01 \uff1e2778 \uff1e500 0.49 \u00b1 0.04 \uff1e1020 \uff1e500 0.59 \u00b1 0.05 \uff1e847\n Hypoxia (1% O2)\n Ru \uff1e112 0.41 \u00b1 0.01 \uff1e273 \uff1e112 0.40 \u00b1 0.01 \uff1e280 \uff1e112 0.42 \u00b1 0.01 \uff1e267\n TiO2@Ru \uff1e500 0.47 \u00b1 0.01 \uff1e1064 \uff1e500 1.16 \u00b1 0.05 \uff1e431 \uff1e500 1.06 \u00b1 0.04 \uff1e472\n TiO2@Ru@siRNA \uff1e500 0.22 \u00b1 0.01 \uff1e2273 \uff1e500 0.58 \u00b1 0.04 \uff1e862 \uff1e500 0.61 \u00b1 0.02 \uff1e820\n a\n The IC50 values are calculated based on the concentration of Ru.\n b\n Cells were incubated with the tested compounds for 48 h and detected by MTT assay in the absence and presence of 525 nm light (15 mW cm\u2212 2, 30 min).\n c\n PI is the ratio of the IC50 value in the dark to that obtained upon light irradiation. Data are presented as the means \u00b1 standard deviations (SD).\n\n\n\n\nFig. 2. Silence of HIF-1\u03b1 by TiO2@Ru@siRNA. (A) Time-dependent colocalization of TiO2@Ru@FAM-siRNA with LTDR in HN6 cells. Cells were treated with\nTiO2@Ru@siRNA (0.1 \u03bcg/mL) under normoxia and imaged by a confocal microscope at different time points in the absence and presence of light (525 nm, 15 mW\ncm\u2212 2, 30 min). LTDR (200 nM) was added 15 min before imaging. Overlay 1: FAM-siRNA and Ru; Overlay 2: FAM-siRNA and LTDR; Overlay 3: Ru and LTDR;\nOverlay 4: FAM-siRNA, Ru and LTDR in bright filed. Ru: \u03bbex = 488 nm; \u03bbem = 630 \u00b1 20 nm. FAM-siRNA: \u03bbex = 488 nm; \u03bbem = 510 \u00b1 20 nm. LTDR: \u03bbex = 633 nm; \u03bbem\n= 720 \u00b1 20 nm. Scale bars: 10 \u03bcm. (B, C) The expression of HIF-1\u03b1 in HN6 cells treated with TiO2@Ru@siRNA (0.1, 0.2, 0.4, 0.8 \u03bcg/mL, 24 h) in the presence of\nlight (525 nm, 15 mW cm\u2212 2, 30 min) was measured by RT-qPCR (B) and western blotting (C) under hypoxia. Quantitative analysis of western blotting was obtained\nto determine the relative intensity of HIF-1\u03b1. **p < 0.01, ****p < 0.0001.\n\n\n3.2. TiO2@Ru@siRNA shows high phototoxicity under normoxia and (Table 1 and Table S1). Both under normoxic and hypoxic conditions,\nhypoxia Ru, TiO2@Ru and TiO2@Ru@siRNA in the absence of light are\n nontoxic on all the cell lines tested. Generally, the phototoxicity follows\n The PDT activities of Ru, TiO2@Ru and TiO2@Ru@siRNA in vitro the order: TiO2@Ru@siRNA > TiO2@Ru > Ru, and HN6 is the most\nwere evaluated on human tongue squamous cell carcinoma (HN6, HSC-6 sensitive cell line to the PDT treatment.\nand HSC-3) and dysplasia oral keratinocyte (DOK) cells by MTT assay Under normoxic condition, the PI of TiO2@Ru@siRNA in HN6 cells\n\n 6\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 3. TiO2@Ru@siRNA-mediated PDT causes pyroptosis through lysosomal damage. (A) Impact of TiO2@Ru@siRNA on lysosomal membrane permeability in\nHN6 cells measured by AO staining and confocal microscopy. The cells were treated with TiO2@Ru@siRNA (0.8 \u03bcg/mL, 24 h) under hypoxia and then irradiated\nwith a 525 nm laser (15 mW cm\u2212 2, 30 min). \u03bbex = 488 nm, \u03bbem = 525 \u00b1 20 nm. Scale bars: 10 \u03bcm. (B) Intracellular ROS levels measured by DCFH-DA staining with\nflow cytometry. HN6 cells were treated with the TiO2@Ru@siRNA (0.4, 0.8 \u03bcg/mL) for 24 h under hypoxia and irradiated with a 525 nm laser (15 mW cm\u2212 2, 30\nmin) before incubated with DCFH-DA (10 \u03bcM, 15 min). \u03bbex = 488 nm, \u03bbem = 525 \u00b1 20 nm. (C) Impact of different ROS scavengers on the cellular ROS level. Cells\nwere pre-incubated with the ROS scavengers for 2 h (Trion: 10 mM; NaN3: 5 mM; D-mannitol: 50 mM; Ebselen: 50 \u03bcM). The cells were treated with TiO2@R\u00ad\nu@siRNA (0.8 \u03bcg/mL, 24 h) under hypoxia and then irradiated with a 525 nm laser (15 mW cm\u2212 2, 30 min). (D) The impact of ROS scavengers (Trion: 10 mM; NaN3:\n5 mM; D-mannitol: 50 mM; Ebselen: 50 \u03bcM) on the cell viability of TiO2@Ru@siRNA. The cells were treated with TiO2@Ru@siRNA (0.25, 0.5, 0.75, 1 \u03bcg/mL, 24 h)\nunder hypoxia and then irradiated with a 525 nm laser (15 mW cm\u2212 2, 30 min). (E) Bright field images by confocal microscope of HN6 cells treated with\nTiO2@Ru@siRNA (0.8 \u03bcg/mL) for 24 h and then irradiated with a 525 nm laser (15 mW cm\u2212 2, 30 min) under hypoxia. Scale bars: 10 \u03bcm. (F, G) The alternations in\ncell morphology upon PDT treatment by TiO2@Ru@siRNA (0.8 \u03bcg/mL, 24 h) combined with a 525 nm laser (15 mW cm\u2212 2, 30 min) detected by SEM (F) and TEM\n(G). Scale bars: 10 \u03bcm (SEM), 5 \u03bcm and 20 \u03bcm (TEM). (H) Western blotting of the impact of PDT by TiO2@Ru@siRNA on the expression of GSDMD and cleaved\ncaspase-1 in HN6 cells. Statistical analysis of western blotting to determine the relative intensity of GSDMD and cleaved caspase-1. The cells were treated with\nTiO2@Ru@siRNA (24 h) at the indicated concentrations under hypoxia and then irradiated with a 525 nm laser (15 mW cm\u2212 2, 30 min). ****p < 0.0001.\n\n\n\n\n 7\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 4. The impact of TiO2@Ru@siRNA-mediated PDT on transcriptome by RNA-seq. (A) Volcano plots showing the DEGs in HN6 cells treated with TiO2@R\u00ad\nu@siRNA-mediated PDT. The cells were treated with TiO2@Ru@siRNA (0.4 \u03bcg/mL, 24 h) under hypoxia + light (525 nm, 15 mW cm\u2212 2, 30 min) and then incubated\nfor another 4 h before mRNA extraction. (B) KEGG enrichment analysis of DEGs after TiO2@Ru@siRNA-mediated PDT treatment. (C) GO term enrichment analysis\nof DEGs including biological process, cellular component, and molecular function was conducted to explain the enriched pathways and functions. (D) GSEA analysis\nof genes in different pathways. (E) RT-qPCR verification of the up-regulation of HSPA1A, HSPA1B and IL-24 mRNA in HN6 cells treated by TiO2@Ru@siRNA (0.4\n\u03bcg/mL, 24 h) under hypoxia with a 525 nm laser (15 mW cm\u2212 2, 30 min). **p < 0.01, ****p < 0.0001.\n\nis about 5-fold higher than that of Ru, and difference in phototoxicity is incubation, TiO2@Ru@FAM-siRNA can be effectively absorbed by HN6\nfound between TiO2@Ru and TiO2@Ru@siRNA. Similarly, phototox\u00ad cells, and a high Pearson\u2019s correlation coefficient (PCC: 0.82) is\nicity of Ru, TiO2@Ru and TiO2@Ru@siRNA is well maintained under observed for it with LTDR. Interestingly, with the extension of time, Ru\nhypoxic condition, and a PI value higher than 2000 is observed for can still form a good colocalization with LTDR, while the colocalization\nTiO2@Ru@siRNA in HN6 cells. These results show that TiO2@R\u00ad of FAM-siRNA with Ru and LTDR is gradually lost. The result indicates\nu@siRNA can overcome the hypoxic microenvironment of tumor, and that siRNA successfully escapes from lysosomes/endosomes. Besides,\nthe high PDT effects of TiO2@Ru@siRNA are attributed to its capability the light irradiation can accelerate the release of FAM-siRNA from\nto simultaneously act through both Type I and II pathways. TiO2@Ru@siRNA NPs, which may be attributed to the lysosomal\n damage by ROS generated by the NPs (Fig. 2A). Additionally, after 24 h\n incubation, most of the siRNA loaded on the nanoparticles has released\n3.3. Silence of HIF-1\u03b1 by TiO2@Ru@siRNA to cytoplasm in the absence of light, which could protect the siRNA from\n the oxidization by the ROS produced in the following PDT process. Be\u00ad\n Considering that nanomaterials are usually retained in lysosomes/ sides, the releasing of HIF-1\u03b1 siRNA could also relieve the hypoxia status\nendosomes through endocytosis, firstly we studied the cellular locali\u00ad in cancer cells favoring for the development of photodynamic effect of\nzation of TiO2@Ru@siRNA in OSCC cells using the commercial dye the TiO2 nanoparticles (Figure S10).\nLTDR. To monitor the release of siRNA, HIF-1\u03b1 siRNA in TiO2@R\u00ad As a consequence, both real-time quantitative polymerase chain\nu@siRNA was labeled with carboxyfluorescein (FAM). After 2 h\n\n 8\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 5. TiO2@Ru@siRNA enhances anticancer immunity. (A) Kaplan\u2013Meier survival curve of overall survival based on head and neck squamous cell carcinoma\npatients with high- and low-expression of IL-24 in TCGA. (B) Representative flow cytometric analysis and relative expression of IFN-\u03b3 expression gated on CD4+ T\ncells and CD8+ T cells stimulated by conditioned medium. HN6 cells were treated with TiO2@Ru@siRNA (0.4 \u03bcg/mL, 24 h) under hypoxia with a 525 nm laser\nirradiation (15 mW cm\u2212 2, 30 min) and then incubated for another 24 h. The conditioned medium was collected and centrifuged at 500 g for 5 min to remove floating\ndead cells and debris. Peripheral blood mononuclear cells (PBMCs) were isolated from OSCC patients and stimulated by conditioned medium (20% and 40% in\ncomplete medium) for 48 h. (C) Flow cytometric analysis for the impact of PDT on the expression of HMGB1. HN6 cells were incubated with TiO2@Ru@siRNA at the\nindicated concentrations (0.2, 0.4, 0.8 \u03bcg/mL) for 24 h and then irradiated with a 525 nm light array (15 mW cm\u2212 2, 30 min) under hypoxia. (D) Impact of PDT on the\nexpression of PD-L1 and NF-\u03baB. Quantitative analysis of western blotting was obtained to determine the relative intensity of PD-L1 and NF-\u03baB. HN6 cells were\nincubated with TiO2@Ru@siRNA at the indicated concentrations (0.1, 0.2, 0.4, 0.8 \u03bcg/mL) for 24 h and then irradiated with a 525 nm light array (15 mW cm\u2212 2, 30\nmin) under hypoxia. (E) Kaplan\u2013Meier survival curve of overall survival based on OSCC patients with high- and low-expression of PD-L1 in GSE41613 dataset. (F)\nSchematic illustration of the mechanisms of cancer immunomodulatory effects by TiO2@Ru@siRNA-mediated PDT. *p < 0.05, **p < 0.01.\n\n\nreaction (RT-qPCR; Fig. 2B) and western blotting results suggest that 3.4. TiO2@Ru@siRNA-mediated PDT causes pyroptosis through\nHIF-1\u03b1 is successfully knocked down under hypoxia in a concentration- lysosomal damage\ndependent manner (Fig. 2C).\n As TiO2@Ru@siRNA localizes in lysosomes, we then evaluated the\n photo-damage of lysosomes by acridine orange (AO) staining upon PDT\n treatment. AO emits red fluorescence in acidic lysosomes and green\n\n\n 9\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\nfluorescence in the cytoplasm and nuclei. The red dots representing janus kinase/signal transduction and activator of transcription (JAK-\nacidic lysosomes are gradually disappeared upon TiO2@Ru@siRNA- STAT) signaling pathway and apoptosis (Fig. 4B). These pathways are\nmediated PDT treatment, indicating the increase in the lysosomal involved in cell proliferation, differentiation and apoptosis [44\u201346].\nmembrane permeability (Fig. 3A). Especially, MAPK and JAK-STAT signaling pathways mediate lysosomal\n The capability of TiO2@Ru@siRNA to generate ROS upon light integrity [47,48].\nirradiation was detected using DCFH-DA staining, which can be oxidized Gene Ontology (GO) enrichment analysis shows that TiO2@R\u00ad\nto the highly emissive 2\u2032 ,7\u2032 -dichlorofluorescein (DCF) by cellular ROS. u@siRNA-mediated PDT mainly influences positive regulation of\nIn the presence of light, a concentration-dependent increase in DCF interferon-gamma (IFN-\u03b3) production, regulation of interleukin-10\nfluorescence is observed for TiO2@Ru and TiO2@Ru@siRNA under biosynthetic process, CD8+ T cell differentiation, regulation of target\nboth normoxia and hypoxia. In the presence of light, TiO2@Ru@siRNA of rapamycin complex 1 (TORC1) signaling and nuclear factor-kappa B\n(0.8 \u03bcg/mL) elevates the cellular ROS level to about 20-fold under both (NF-\u03baB) complex (Fig. 4C). These pathways are closely associated with\nhypoxia and normoxia (Fig. 3B and Figure S11). cancer immunity [49\u201352]. Consistent with the fact that TiO2@R\u00ad\n To confirm which kind of ROS played a major role in TiO2@R\u00ad u@siRNA can photo-damage lysosomes, the significantly regulated\nu@siRNA- and TiO2@Ru-mediated PDT, the effects of different ROS TORC1 signaling is an important regulator of lysosomes, participating in\nscavengers (D-mannitol: \u2022OH; Tiron: \u2022O\u22122 ; Sodium azide (NaN3): 1O2; lysosomal activation and regulating cell growth and metabolism [53].\nEbselen: ONOO\u2212 ) on the oxidative stress caused by the nanocomposites As expected, gene set enrichment analysis (GSEA) shows that\nwere investigated. Tiron, NaN3 and mannitol effectively suppress the response to ROS, regulation to cell death and immune response related\nproduction of ROS by TiO2@Ru@siRNA and TiO2@Ru under both genes are upregulated after PDT treatment by TiO2@Ru@siRNA\nhypoxia and normoxia, while ebselen can only slightly reduce the ROS (Fig. 4D). Several immune-related genes are found to be significantly\nlevel. These results suggest that TiO2@Ru@siRNA and TiO2@Ru upregulated upon PDT treatment by TiO2@Ru@siRNA including\nmainly produce \u2022O\u22122 , 1O2 and \u2022OH to kill tumor cells upon PDT under interleukin-24 (IL-24) and heat shock protein family A (HSPA1A/\nhypoxia and normoxia (Fig. 3C and Figure S12). Moreover, Tiron, NaN3 HSPA1B) closely associated with cancer immune microenvironment\nand mannitol markedly increase the viability of HN6 cells under hypoxia (Fig. 4A).\n(Fig. 3D), while ebselen only slightly increases the viability of HN6 cells Notably, IL-24, an immunomodulatory and tumor suppressor gene\nsubjected to PDT treatment by TiO2@Ru@siRNA, which further elab\u00ad which is suppressed in many types of cancer [54], is significantly\norates that \u2022O\u22122 , 1O2 and \u2022OH account for the cell killing effect of PDT upregulated upon PDT treatment by TiO2@Ru@siRNA. An increased\nunder hypoxia. expression of HSPA1A and HSPA1B, which can induce CD8+ cytotoxic T\n The alternations in cell morphology upon TiO2@Ru@siRNA treat\u00ad lymphocyte and CD4+ T helper cell responses [55], is also detected in\nment in combination with light were detected by confocal microscopy, TiO2@Ru@siRNA-treated samples in the presence of light. The eleva\u00ad\nscanning electron microscopy (SEM) and TEM. Confocal images show tion in the transcription of HSPA1A, HSPA1B and IL-24 in samples with\nthat TiO2@Ru@siRNA-mediated PDT causes swollen cells with large PDT is also verified by RT-qPCR (Fig. 4E).\nbubbles protruding from the plasma membrane under hypoxia and\nnormoxia (Fig. 3E and Figure S13). SEM shows that control cells have 3.6. TiO2@Ru@siRNA enhances anticancer immunity\nclear outlines, long protuberances and tight cell connections, while cells\ntreated with TiO2@Ru@siRNA in combination with light display As RNA-seq shows that TiO2@Ru@siRNA-mediated PDT can influ\u00ad\ntypical ultrastructural characteristics of pyroptosis including cell ence immune-related pathways, we then evaluate the effects of its\nswelling, membrane rupture and reduction of numerous surface villi capability to activate antitumor immunity. As previously indicated, IL-\n(Fig. 3F). TEM shows that cells treated with TiO2@Ru@siRNA plus light 24, also known as melanoma differentiation-associated gene-7 (mda-7)\nappear complete loss of identifiable organelles and large disruption with [56], is upregulated upon PDT treatment. Kaplan\u2013Meier analysis in\u00ad\nnumerous pores in the plasma membrane, in contrast to an intact plasma dicates that lower IL-24 expression predicts poorer prognosis for head\nmembrane and well-defined organelles from the control cells (Fig. 3G). and neck squamous cell carcinoma patients in the TCGA database\nMoreover, the key proteins of pyroptosis, gasdermin D (GSDMD) and (Fig. 5A).\ncleaved caspase-1 [43], are upregulated upon PDT treatment with As shown in Fig. 5B, conditioned medium using the supernatants of\nTiO2@Ru@siRNA under both hypoxia and normoxia, which indicates HN6 cells treated with TiO2@Ru@siRNA-mediated PDT can stimulate\nTiO2@Ru@siRNA-mediated PDT induces caspase-1-dependent canon\u00ad the IFN-\u03b3 expression in both CD4+ and CD8+ T cells derived from the\nical pathway of pyroptosis (Fig. 3H and Figure S14). All these results peripheral blood of OSCC patients compared with control group, which\nshow that TiO2@Ru@siRNA-mediated PDT causes pyroptosis through suggests that PDT treatment may enhance anti-tumor immunity effi\u00ad\nlysosomal damage. ciently in tumor microenvironment. To address whether IL-24 released\n by OSCC cells can affect antitumor immune response of OSCC as well,\n3.5. RNA-seq analysis we examined the effects of exogenous IL-24 on cytokine expression. The\n results suggest that exogenous IL-24 significantly increases IFN-\u03b3 pro\u00ad\n RNA-seq was further performed to investigate the impact of duction in CD4+ and CD8+ T cells (Figure S17).\nTiO2@Ru@siRNA-mediated PDT on transcriptome. The correlation Moreover, the expression of high-mobility group box 1 protein\ncoefficients between every two individual samples from the same group (HMGB1) that can be induced by pyroptosis [57,58], exhibits a\nare above 0.9 (Figure S15), indicating that the RNA-seq experiment is concentration-dependent decrease upon TiO2@Ru@siRNA-mediated\nreproducible. The overall Q30% is above 92.33% (Table S2). More than PDT under hypoxia (Fig. 5C). However, calreticulin (CRT) expression\n92.97% of readings are mapped to reference genes in all samples, and shows no significant change (Figure S18). These results suggest that\n84.53% of readings are located in exons (Table S3 and Figure S16). TiO2@Ru@siRNA-mediated PDT does not induce typical immunogenic\nCompared with the control group, 210 significantly differentially cell death (ICD). The upregulation of IL-24 may be caused by the\nexpressed genes (DEGs; |Fold change| \u2265 2; False discovery rate \u22640.05) downregulation of HMGB1 induced by pyroptosis, as it has been re\u00ad\nare detected, of which 147 genes are significantly up-regulated and 63 ported that knockdown of HMGB1 can induce the upregulation of IL-24\ngenes down-regulated (Fig. 4A). [59].\n Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment The decreased expression of HIF-1\u03b1 may downregulate PD-L1 [60],\nanalysis shows that DEGs caused by TiO2@Ru@siRNA-mediated PDT and the blockade of PD-L1 is effective in clinical cancer treatment [61].\nare enriched in mitogen-activated protein kinase (MAPK) signaling Interestingly, TiO2@Ru@siRNA-mediated PDT can decrease the\npathway, pathway in cancer, cytokine-cytokine receptor interaction, expression of PD-L1. It has been reported that HMGB1-activated NF-\u03baB\n\n 10\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 6. TiO2@Ru@siRNA-mediated PDT exhibits potent antitumor effects in PDX model. (A) Schematic illustration of the therapeutic protocol in PDX model. (B)\nPlots of the changes in tumor volumes during the treatment. (C, D) Representative images of tumor-bearing mice (C) and resected tumors (D) at the end of the\ntreatment. (E) The tumor weight at the end of the treatment. (F) Representative H&E and IHC staining of Ki67 and HIF-1\u03b1 in tumor sections from different groups.\nScale bars: 50 \u03bcm **p < 0.01, ****p < 0.0001.\n\n\ncontributes to cancer immune suppression [62]. Accordingly, the 3.7. TiO2@Ru@siRNA inhibits OSCC in PDX model\nexpression of NF-\u03baB is also downregulated after PDT under hypoxia\n(Fig. 5D). PDX, in which tumor fragments surgically dissected from cancer\n Meanwhile, Kaplan\u2013Meier analysis indicates that significantly patients are transplanted into immunodeficient mice, has emerged as a\nhigher PD-L1 expression predicts poorer prognosis for OSCC patients in useful model for translational research to facilitate precision medicine\nGSE41613 and GSE65858 datasets (Fig. 5E and Figure S1). These results [63]. PDX models reflect the diversity and heterogeneity of tumors, so\nindicate that the TiO2@Ru@siRNA-mediated PDT can downregulate the susceptibility of PDX to anticancer treatment is closely correlated\nHMGB1 and NF-\u03baB, which further inhibits PD-L1 to alleviate the cancer with clinical data in patients [64]. Importantly, PDX can maintain the\nimmunosuppression. Decreased expression of HMGB1 can also induce cellular and histopathological structure of their parental tumors.\nIL-24 upregulation to activate CD4+ and CD8+ T cells with IFN-\u03b3 Therefore, PDX is a promising model in predicting the efficacy of con\u00ad\nsecretion for enhanced anticancer immune responses. Therefore, the ventional and novel anti-cancer therapies [65,66]. A PDX model was\nimmunomodulatory effects induced by TiO2@Ru@siRNA-mediated established in BALB/c mice (female, 4 weeks) to evaluate the antitumor\nPDT may have a positive effect on cancer treatment (Fig. 5F). effects of TiO2@Ru@siRNA in vivo (Fig. 6A).\n TiO2@Ru@siRNA-medidated PDT shows a remarkable inhibition\n on tumor growth (Fig. 6B, C and D). At the end of the treatment, the\n\n\n 11\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\n\n\nFig. 7. TiO2@Ru@siRNA inhibits carcinogenesis in 4NQO induced rat model. (A) Schematic overview of the 4NQO model experimental design. (B) Representative\nimages of rat tongue lesions in the four groups at 17 week and 21 week. (C) Representative gross observation and H&E images of the rat tongues in different groups at\nendpoint. Scale bars: 200 \u03bcm. (D) H&E scores of the histopathologic diagnoses in the four groups. (E) Quantification of the histological ratio of low-grade dysplasia\n(normal and mild dysplasia), high-grade dysplasia (moderate and severe dysplasia), and carcinoma tissues in the four groups. (F) Representative IHC staining of Ki67,\nHIF-1\u03b1, CD3 and GSDMD. Scale bars: 50 \u03bcm *p < 0.05.\n\n\n\n\n 12\n\fJ.-Y. Zhou et al. Biomaterials 289 (2022) 121757\n\n\ntumor weight decreases by approximately 10-fold for TiO2@Ru@siRNA carcinogenesis models. In conclusion, we have constructed a hypoxia-\ngroup with PDT (Fig. 6E). H&E staining exhibits more necrotic foci in adaptive photoimmunotherapeutic nanosystem for OSCC therapy, and\nthe TiO2@Ru@siRNA-mediated PDT group. The nuclear antigen Ki67 is its possibility of potential clinical transformation is still under\nstrongly associated with tumor cell proliferation and growth, and is exploration.\nwidely estimated in the immunohistochemical (IHC) staining as a tumor\nproliferation marker. Consistently, IHC results show that the expression Credit author statement\nof Ki67 and HIF-1\u03b1 is significantly decreased in TiO2@Ru@siRNA-\ntreated mice both in the absence and presence of light with the PDT Jia-Ying Zhou: Data curation, Methodology, Formal analysis,\ngroup exerting a more profound effect (Fig. 6F). Investigation, Software, Writing \u2013 original draft. Wen-Jin Wang: Visu\u00ad\n Furthermore, no death or apparent decrease in body weights is found alization, Methodology, Validation, Writing \u2013 original draft. Chen-Yu\nin all the groups (Figure S19), and H&E staining of the major organs Zhang: Visualization, Methodology, Software, Validation, Writing \u2013\nreveals no noticeable organ damage, which indicates that TiO2@R\u00ad original draft. Yu-Yi Ling: Data curation, Methodology, Validation.\nu@siRNA NPs possess no systematic toxicity (Figure S20). Xiao-Jing Hong: Visualization, Resources, Validation. Qiao Su: Meth\u00ad\n odology, Validation. Wu-Guo Li: Methodology, Validation. Zong-Wan\n3.8. TiO2@Ru@siRNA inhibits carcinogenesis in 4NQO induced rat Mao: Methodology, Resources, Supervision. Bin Cheng: Methodology,\nmodel Funding acquisition, Supervision. Cai-Ping Tan: Conceptualization,\n Formal analysis, Funding acquisition, Supervision, Writing \u2013 review &\n The development of OSCC is a multistep and dynamic process, which editing. Tong Wu: Conceptualization, Project administration, Funding\nincludes developing stages of hyperplasia, dysplasia, carcinoma in situ acquisition, Supervision, Writing \u2013 review & editing.\nand finally the invasive carcinoma [67]. 4NQO is a carcinogen known to\ninduce DNA damage, leading to premalignant and malignant lesions in\nthe oral cavity, which is similar to histological and molecular changes Declaration of competing interest\nobserved in human oral carcinogenesis [68]. Thus, 4NQO model pro\u00ad\nvides an excellent opportunity to evaluate the capability of the anti\u00ad The authors declare that they have no known competing financial\ncancer treatment to intervene epithelial malignant transformation and interests or personal relationships that could have appeared to influence\ntumor progression [69]. Therefore, after confirming the antitumor effect the work reported in this paper.\nof TiO2@Ru@siRNA-mediated PDT in PDX model, its potency to inhibit\nthe carcinogenesis of 4NQO-induced rat model was further evaluated. Data availability\n The schematic diagram of 4NQO rat model construction and thera\u00ad\npeutic protocol is shown in Fig. 7A. After 4NQO exposure for 16 weeks, Data will be made available on request.\nthe lesions show granular hyperplasia, white flakes and spot plaques. At\nthe end of the intervention, the lesions in the control group are Acknowledgements\naccompanied by erosion, ulcer, and endogenous growth with unclear\nboundaries, while the lesions in TiO2@Ru@siRNA-mediated PDT group This study was supported by the Science and Technology Planning\nstill exhibit plaques (Fig. 7B). TiO2@Ru@siRNA-treated mice in com\u00ad Project of Guangzhou, China (grant number 202206080009), the Sci\u00ad\nbination with light significantly interrupts the malignant transformation ence and Technology Planning Project of Guangdong Province, China\nprocess. A histological comparison shows that the mean score of the (grant number 2018B030317001), the National Natural Science Foun\u00ad\nTiO2@Ru@siRNA group with PDT (HE score = 3.6) is significantly dation of China (grant numbers 22022707, 22177142 and 21837006)\nlower than that in the control group (HE score = 7.2; p < 0.05; Fig. 7C and the Fundamental Research Funds for the Central Universities. J.Y.\nand D). Zhou, W.J. Wang, C.Y. Zhang and Y.Y. Ling contributed equally to this\n Furthermore, the proportion of samples with different malignant work.\ndegree was calculated within each group. In the TiO2@Ru@siRNA +\nPDT group, only 1 (20%) sample develops into carcinoma in situ and 1 Appendix A. Supplementary data\n(20%) sample develops into mild invasion carcinoma. While in control\nand control + PDT groups, 4 (80%) samples develop into invasion car\u00ad Supplementary data to this article can be found online at https://doi.\ncinoma (Fig. 7E and Table S4). IHC results show that the TiO2@R\u00ad org/10.1016/j.biomaterials.2022.121757.\nu@siRNA + PDT group significantly decreases the positive rates of Ki67\nas well as HIF-1\u03b1 and increases the expression of CD3 and GSDMD in References\ntumors (Fig. 7F). Taken together, these results indicate that the\nTiO2@Ru@siRNA-mediated PDT exhibits significant antitumor effects [1] B.W. Neville, T.A. Day, Oral cancer and precancerous lesions, CA A Cancer J. Clin.\non OSCC in vivo. In particular, blocking the malignant transformation 52 (2002) 195\u2013215.\n [2] S.B. Chinn, J.N. 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