Self-assembly of a ruthenium-based cGAS-STING photoactivator for carrier-free cancer immunotherapy.

{"full_text": " European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\n Contents lists available at ScienceDirect\n\n\n European Journal of Medicinal Chemistry\n journal homepage: www.elsevier.com/locate/ejmech\n\n\nResearch paper\n\nSelf-assembly of a ruthenium-based cGAS-STING photoactivator for\ncarrier-free cancer immunotherapy\nYu-Yi Ling a, b, 1 , Zhi-Yuan Li a, b, 1 , Xia Mu c, 1 , Ya-Jie Kong a, b , Liang Hao a, b , Wen-Jin Wang a, b ,\nQing-Hua Shen a, b , Yue-Bin Zhang c, ** , Cai-Ping Tan a, b, *\na\n MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, Sun Yat-Sen University, Guangzhou, 510006, PR China\nb\n Guangdong Basic Research Center of Excellence for Functional Molecular Engineering, Guangzhou, 510006, PR China\nc\n State Key Laboratory of Molecular Reaction, Dynamics, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, 116023, 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\nHandling Editor: Dr. Z Liu The cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) pathway promotes antitumor\n immune responses by sensing cytosolic DNA fragments leaked from nucleus and mitochondria. Herein, we\nKeywords: designed a highly charged ruthenium photosensitizer (Ru1) with a \u03b2-carboline alkaloid derivative as the ligand\ncGAS-STING for photo-activating of the cGAS-STING pathway. Due to the formation of multiple non-covalent intermolecular\nSelf-assembly\n interactions, Ru1 can self-assemble into carrier-free nanoparticles (NPs). By incorporating the triphenylphos\u00ad\nRu(II) complexes\n phine substituents, Ru1 can target and photo-damage mitochondrial DNA (mtDNA) to cause the cytoplasmic\nDNA binding\nPhotoimmunotherapy DNA leakage to activate the cGAS-STING pathway. Finally, Ru1 NPs show potent antitumor effects and elicit\n intense immune responses in vivo. In conclusion, we report the first self-assembling mtDNA-targeted photosen\u00ad\n sitizer, which can effectively activate the cGAS-STING pathway, thus providing innovations for the design of new\n photo-immunotherapeutic agents.\n\n\n\n\n1. Introduction The activation of cGAS-STING cascade can release immune-\n stimulatory factors to attenuate tumour growth and recruit immune\n Since 2008, Barber et al. identify STING (stimulator of interaction cells for tumour suppression [13,14]. Moreover, STING-IRF3-induced\ngenes) as a vital component of host innate immunity, it is found to show type I interferon production can induce tumour antigen presentation\nsubstantial capacity in virus/bacterial/parasitic infections and cancer on dendritic cells and macrophages for cross-priming of CD8+ T cells\nimmunity by regulating protein synthesis and interferon (IFN) expres\u00ad [15,16]. The cGAS-STING pathway can be activated by cytosolic dsDNA\nsion [1,2]. Double-stranded DNA (dsDNA) induces the activation of with exogenous origins or endogenous genomic and mitochondrial DNA\ncyclic GMP-AMP synthase (cGAS) [3\u20136]. Upon binding dsDNA, the (mtDNA) [17,18]. Multiple strategies have been developed to activate\ndimerization of cGAS leads to its enzyme activation and the synthesis of the STING pathway in tumour cells or tumour infiltrating immune cells\n2\u2032-3\u2032-cyclic GMP-AMP (cGAMP) [7,8]. cGAMP binds to the dimer of [19,20]. Based on the typical molecular mechanisms, STING agonists or\nSTING located on the endoplasmic reticulum (ER) membrane, leading to nanocarriers, as well as induction of mitochondrial and nuclear dsDNA\nconformational changes and oligomerization of STING [3,9]. Then, release, can activate the cGAS-STING signaling pathway [21]. Most of\nSTING promotes the autophosphorylation of TANK binding kinase 1 the agents currently in clinical trials are cyclic dinucleotide (CDN) de\u00ad\n(TBK1) and recruit interferon regulatory factor 3 (IRF3) [10,11]. The rivatives mimicking cGAMP, which are hindered by their high molecular\nTBK1 phosphorylates IRF3, which leads to the dimerization and trans\u00ad weight, poor membrane permeability, and poor stability [22]. Non-CDN\nlocation of IRF3 to the nucleus to induce the expression of type I IFN, agonists, e.g., diABZI-Compound 3 from GSK [23], SR717 from Scripps\nInterferon-stimulated gene (ISG) and several other inflammatory me\u00ad Research [24], MSA-2 from Merck [25], and NVS-STG2 from Novartis\ndiators and chemokines [12]. [26], exhibit encouraging efficacy in vitro and in vivo. However, the\n\n\n\n * Corresponding author. MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, Sun Yat-Sen University, Guangzhou 510006, PR China.\n ** Corresponding author.\n E-mail addresses: zhangyb@dicp.ac.cn (Y.-B. Zhang), tancaip@mail.sysu.edu.cn (C.-P. Tan).\n 1\n These authors contributed equally to this work.\n\nhttps://doi.org/10.1016/j.ejmech.2024.116638\nReceived 4 May 2024; Received in revised form 21 June 2024; Accepted 27 June 2024\nAvailable online 28 June 2024\n0223-5234/\u00a9 2024 Elsevier Masson SAS. All rights are reserved, including those for text and data mining, AI training, and similar technologies.\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\nsystemic administration of STING agonists may induce \u201ccytokine storm\u201d a strategy for designing self-assembled cGAS-STING activators, which\nto damage normal tissues, which limits their further clinical use [17], To may provide ideas for the design of novel photo-immunotherapeutic\nresolve these issues, the tumour-targeted delivery agents of STING ag\u00ad agents.\nonists, e.g., nanoscale coordination polymers [27], polymer-based\nplatform [28\u201330], lanthanide-nucleotide coordination nanoparticles 2. Results and discussion\n[31] and nucleotide nanocomplex-decorated ultrasound microbubbles\n[32,33], have been developed. 2.1. Synthesis and characterization\n Compared with traditional cancer therapy, photodynamic therapy\n(PDT) has attracted extensive attention as it is non-invasive [34\u201336]. Ru1 was synthesized by reacting the precursor cis-[Ru(DMSO)2Cl2]\nPDT uses photosensitizers (PSs) to transfer energy from light to oxygen (DMSO = Dimethyl sulfoxide) with 1 equiv of 1-(2-pyridyl)-\u03b2-carboline)\nin the tumour, thus producing highly cytotoxic reactive oxygen species (1-Py-\u03b2C) and 2 equiv of (6-(1,10-phenanthroline-5-carboxamido)\n(ROS), leading to tumour tissue necrosis or apoptosis [35]. Among the hexyl)triphenylphosphonium (phen-PPh3) at 140 \u25e6 C for 6 h, followed\ntransition metal-based photosensitizers studied at present, Ru(II) poly\u00ad by purification by silica chromatography (Scheme S1, Supporting In\u00ad\npyridine complexes are the most attractive candidates, which can be formation). The control compound Ru2 was synthesized by reacting the\nascribed to their long excited state lifetimes, effective 1O2 sensitization, precursor cis-[Ru(phen)2Cl2] (phen = 1,10-phenanthroline) with the\nvisible light absorption, high cell penetration capability and ligand 1-Py-\u03b2C and purified by silica chromatography as previously\ntwo/multi-photon absorptions [37,38]. In particular, the Ru(II) based described [52]. Ru1 and Ru2 were characterized by ESI-MS, 1H NMR\nphotosensitizer TLD1433 developed by McFarland and her colleagues spectroscopy, 13C NMR spectroscopy and HPLC (Figs. S1\u2013S11).\nhas entered clinical trials for bladder cancer treatment [39]. Because PSs The absorption spectra of Ru1 and Ru2 are characterized by intense\nare not easy to accumulate in tumor tissues, it is difficult for PSs to spin-allowed intraligand (1IL) absorption bands in the UV region at ca.\naccurately target cancer cells [40]. A variety of nanomaterials (including 250\u2013340 nm and less intense spin-allowed metal-to-ligand charge\npeptides and polymer/inorganic materials) have been used for PS de\u00ad transfer (1MLCT) absorption bands at ca. 350\u2013530 nm, which are typical\nlivery to enhance tumor targeting and blood circulation time [41\u201343]. absorption properties of Ru(II) polypyridine complexes (Fig. 1A and\nDue to the enhanced permeability and retention (EPR) effect, the S12) [52]. Ru1 and Ru2 show strong fluorescence emission when\nnanoparticles can accumulate in tumor tissues to improve the accuracy excited at 450 nm, and the maximum emission peak is about 620 nm\nand efficiency of PDT and minimize the off-targeted effects [44]. How\u00ad (Fig. 1B and S13). The emission quantum yields of Ru1 and Ru2 in\never, the nanocarriers also have many disadvantages, including complex phosphate buffered saline (PBS), CH3CN and CH2Cl2 fall in the range\nmanufacturing, poor stability after dilution, limited drug loading, pre\u00ad between 0.038 and 0.077 (Table S1). UV/vis spectroscopy monitoring\nmature drug release and potential toxicity [45]. In recent years, carrier shows that Ru1 and Ru2 are stable upon irradiation with a 425 nm laser\nfree self-assembled nanomedicine/photosensitizers constructed through for 30 min in PBS (pH 7.4, Fig. S14). The quantum yields of Ru1 and\ntuning of non-covalent intermolecular interactions have attracted Ru2 to photosensitize the generation of singlet oxygen (1O2) are (0.274\nattention due to the ease of preparation and less susceptibility to un\u00ad \u00b1 0.022) and (0.163 \u00b1 0.006), respectively (Fig. 1B). Upon the addition\nexpected side effects [46\u201349]. of sodium azide (NaN3), a known 1O2 quencher, the quantum yields of\n Previously, we found that mtDNA damage can stimulate the cGAS- 1\n O2 for Ru1 and Ru2 were decreased to (0.160 \u00b1 0.006) and (0.074 \u00b1\nSTING pathway [50,51]. Considering the carrier-free strategy based 0.012), respectively, further indicating that Ru1 has the stronger\non self-assembly is helpful to avoid the toxicity of the carrier, and photodynamic effect than Ru2.\nphoto-induced mtDNA damage can achieve the temporal and spatial\ncontrol of cGAS-STING activation, we designed a ruthenium complex\n 2.2. Self-assembly of Ru1 enhances the cellular uptake efficacy and PDT\n(Ru1) containing two ancillary ligands with triphenylphosphine modi\u00ad\n activities\nfied alkyl chains and a main ligand of a bioactive \u03b2-carboline alkaloid\nderivative (Scheme 1). As it can form multivalent intermolecular in\u00ad\n The PDT activities of Ru1 and Ru2 in vitro were evaluated in human\nteractions, Ru1 can self-assemble into stable nanoparticles. Due to the\n triple negative breast cancer (TNBC) MDA-MB-231 cells, mouse TNBC\npresence of the highly charged triphenylphosphine groups, Ru1 can\n 4T1 cells, human normal breast epithelial MCF-10 A cells and human\ntarget mitochondria. Upon light irradiation, Ru1 can damage mtDNA to\n cervical cancer Hela cells (Table 1 and S2). With IC50 values being\ncause cytoplasmic dsDNA release for the activation of the cGAS-STING\n higher than 50 \u03bcM, both Ru1 and Ru2 can be considered to be nontoxic\npathway both in vitro and in vivo (Scheme 1). In summary, we propose\n in dark. In the presence of light, the IC50 values of Ru1 drop to about 0.5\n\n\n\n\nScheme 1. Action mechanisms of Ru1 and chemical structures of Ru1 and Ru2. Ru1 can self-assemble into carrier-free NPs, penetrate into cancer cells through\nendocytosis, and effectively target mitochondrial and reside in tumour tissue. Upon light irradiation, Ru1 can damage mtDNA, leading to the cytoplasmic DNA\nrelease, thereby activating the cGAS-STING pathway and causing dendritic cells (DCs) maturation and T cell activation. Created with BioRender.com.\n\n 2\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\n\n\nFig. 1. (A) UV/Vis absorption spectra and emission spectra of Ru1/Ru2 (20 \u03bcM) measured in degassed PBS buffer (pH 7.4, with 1 % DMSO) at 298 K. \u03bbex = 450 nm.\n(B) The capability of Ru1/Ru2 (20 \u03bcM) to photosensitize the generation of 1O2 probed by 9,10-anthracenediyl-bis(methylene) dimalonic acid (ABDA, 100 \u03bcM) in\ndegassed PBS buffer (pH 7.4, with 1 % DMSO) at 298 K, using NaN3 (5 mM) as the 1O2 quencher and [Ru(bpy)3]Cl2 (bpy: 2,2\u2032-bipyridine) as the standard.\n\n\n \u00c5, the cluster is stabilized via the p-\u03c0 interactions between the \u03b2-car\u00ad\nTable 1\n boline group and the alkyl chain. The largest peak is located at 13.3 \u00c5,\nIC50 (\u03bcM, 48 h) values of Ru1 and Ru2 towards TNBC cell lines.\n where the cluster is maintained via both \u03c0-\u03c0 stacking interactions of two\n Compounds MDA-MB-231 4T1 \u03b2-carboline groups and p-\u03c0 interactions between \u03b2-carboline and alkyl\n Dark Light PI Dark Light PI chain, indicating the indispensable role of the \u03b2-carboline group for self-\n Ru1 >50 0.5 \u00b1 >100 36.9 \u00b1 2.7 \u00b1 0.5 13.7\n assembly.\n 0.05 2.1 The formation of self-assembled nanoparticles may enhance the\n Ru2 >50 1.0 \u00b1 >50 >50 33.9 \u00b1 >1.5 cellular uptake efficacy and the tumor penetration and retention effects\n 0.08 2.9 [46,49]. ICP-MS experiment shows that during an incubation period of\n Cisplatin 11.2 \u00b1 8.7 \u00b1 0.7 1.29 13.5 \u00b1 13.6 \u00b1 1.0\n 6 h, the cellular content of Ru1 gradually increases, and its accumula\u00ad\n 0.8 2.6 1.8\n tion in mitochondria is more pronounced compared to that in cytoplasm\na\n Data are presented as the mean \u00b1 standard deviation (SD), and cells were (Fig. 2E). In contrast, the cellular uptake level and the concentration in\nincubated with the tested compounds for 48 h and detected by MTT assay. mitochondria of Ru2 cells are significantly lower than those of Ru1. In\nb\n Cells were incubated with the compounds for 24 h in the dark and then irra\u00ad\n order to further verify that the higher cellular uptake level of Ru1 is due\ndiated with a 425 nm laser (12 J cm\u2212 2).\nc to the formation of NPs, we studied the cellular uptake mechanism of\n PI is the ratio of the IC50 value in the dark to that obtained upon light\nirradiation. Ru1 and Ru2 using different inhibitors [50]. After being pretreated with\n the endocytosis inhibitor chloroquine (CQ), the amount of ruthenium in\n MDA-MB-231 cells is decreased distinctly, while being incubated at 4 \u25e6 C\n\u03bcM and 2.7 \u03bcM in MDA-MB-231 and 4T1 cells, respectively. The pho\u00ad\n doesn\u2019t significantly affect the cellular ruthenium contents (Fig. 2F). The\ntocytotoxicity index (PI) values of Ru1 is higher than Ru2, especially in\n uptake of Ru1 is energy-dependent endocytosis, mainly mediated by\n4T1 cells. As expected, light irradiation doesn\u2019t show profound effects\n clathrin, while the uptake of Ru2 is energy independent passive diffu\u00ad\non the antiproliferative activities of cisplatin. Meanwhile, Ru1 and Ru2\n sion according to our previous reports [52]. Meanwhile, being incubated\nshow relatively lower photocytotoxicities in MCF-10 A cells and HeLa\n at 4 \u25e6 C or pretreated with CQ, the amount of ruthenium in MCF-10 A\ncells (Table S2), implying their selective antitumor effects towards TNBC\n cells is decreased markedly (Fig. S16), which indicates that Ru1 pene\u00ad\ncells.\n trates into normal cells through energy-dependent endocytosis.\n Interestingly, Ru1 can self-assemble into nanoparticles in PBS, while\nRu2 cannot (Fig. 2A). Transmission electron microscopy (TEM) shows\nthat Ru1 forms nanoparticles with a uniform spherical morphology and\n 2.3. Ru1 can induce the aggregation of dsDNA\nan average size of around 30 nm (Fig. 2A), which is smaller than that\ndetected by dynamic light scattering (DLS; about 125 nm; Fig. 2B).\n Since \u03b2-carboline derivatives have been proven to be effective DNA\n Molecular dynamics (MD) simulations were further conducted to\n binding agent [53], and our previous work has proved that Ru(II)\ninvestigate the mechanism of self-assembly of Ru1 molecules into\n complexes with triphenylphosphine-substituted ligands can induce\nnanoparticles. We constructed an initial MD simulation system consist\u00ad\n changes in the aggregation states of DNA [54], we then studied the\ning of 72 Ru1 molecules (6 \u00d7 6 \u00d7 2) with a spacing of 20 \u00c5 (Fig. S15 A).\n binding properties of Ru1 with DNA. The absorbance of Ru1 at about\nIn addition, a comparison system with the presence of 30 NaH2PO4\n 260 nm decreases, and the absorbance around 470 nm increases, which\nmolecules was also built to mimic the PBS buffer used in our experi\u00ad\n suggests that Ru1 may induce DNA aggregation [55]. The calculated\nmental study (Fig. S15 B). Four independent MD simulations of each\n binding constant of Ru1 towards calf thymus-DNA (CT-DNA) obtained\nsystem were performed lasting for 1500 ns and the MD trajectories from\n by UV\u2013vis titration is 1.22 \u00d7 106 M\u2212 1 (Fig. 3A). In the presence of\n500\u20131500 ns were used for analysis. The radial distribution functions\n CT-DNA, the emission intensity of Ru1 is slightly increased by 1.5-fold\n(RDFs) for the distance between Ru\u2013Ru atoms were utilized to evaluate\n (Fig. 3B). The calculated binding constant of Ru1 towards CT-DNA ob\u00ad\nthe degree of aggregation (Fig. 2C). In the absence of NaH2PO4 mole\u00ad\n tained by fluorescence titration is 3.63 \u00d7 105 M\u2212 1. The circular di\u00ad\ncules, the RDF exhibits two peaks at 9.0 \u00c5 and 12.0 \u00c5, respectively. In\n chroism (CD) spectroscopy of CT-DNA incubated with Ru1 show an\nsharp contrast, the presence of NaH2PO4 molecules significantly\n obvious increase in the molar ellipticity of positive bands around 275\nenhanced the aggregation. Three prominent peaks would be easily\n nm, which can be attributed to the base pair stacking, indicating that\nidentified around 9.2 \u00c5, 11.2 \u00c5, and 13.3 \u00c5. A representative snapshot\n intercalative binding mode of Ru1 with CT-DNA alters secondary\nillustrates the spatial geometry corresponding to the three peaks\n structure of duplex DNA [56]. The blue shift of positive bands and the\n(Fig. 2D). The RDF at 9.2 \u00c5 demonstrates \u03c0-\u03c0 conjugation interactions\n appearance of the negative band at 280 nm imply a significant change in\nbetween the phenanthroline groups of two Ru1 molecules, while at 11.2\n CT-DNA conformation [57]. Consistent with our previous findings [54],\n\n 3\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\n\n\nFig. 2. Ru1 can self-assemble into nanoparticles and penetrate into cancer cells by endocytosis. (A) TEM images of the NPs formed by Ru1 in PBS buffer (pH 7.4).\nScale bar: 50 nm. (B) DLS analysis of the NPs formed by Ru1 in PBS buffer (pH 7.4). (C) Radial distribution functions for the distances between Ru\u2013Ru atoms with\n(red line)/without (black line) NaH2PO4 molecules. (D) A representative snapshot to illustrate the key interactions within the cluster. (E) Content of Ru per milligram\nof protein and their intracellular compartment distribution in MDA-MB-231 cells. The cells were incubated with Ru1/Ru2 (20 \u03bcM) for 6 h. (F) Content of Ru per\nmilligram of protein and their intracellular compartment distribution in MDA-MB-231 cells. The cells were incubated with Ru1 (20 \u03bcM) and different inhibitors.\nError bars: S.D., n = 3. *p < 0.05, **p < 0.01, ***p < 0.001.(For interpretation of the references to colour in this figure legend, the reader is referred to the web\nversion of this article.)\n\n\nthe highly charged Ru1 complexes can induce DNA aggregation under distribution analysis using the last 500 ns of MD trajectories from the\ndark conditions (Fig. 3D and S17). At higher concentrations (>15 \u03bcM), clustering profiles clearly shows an enhancement of aggregation pro\u00ad\nthe negative charge on DNA is neutralized by Ru1, and the Ru1-DNA pensity after the insertion of Ru1. Especially, one of our simulated MD\ncomplex moves towards the negative electrode. In the presence of light, trajectories of the AT-CGd-CGm-DNA system manifests that all DNA\nRu1 can efficiently cleavage DNA from supercoiled to nicked form at a segments are connected to each other and form a single large cluster\nconcentration of 1 \u03bcM (Fig. 3E and S17). after 500 ns MD simulations, suggesting an additive effect of Ru1\n MD simulations were employed to further understand the molecular insertion to induce DNA aggregation (Fig. 3F and S23).\nbasis responsible for the aggregation of DNA segments induced by Ru1. In the DNA-only MD simulation system, the interactions between\nThree single insertions of Ru1 at different positions were built for in- DNA segments are predominantly contributed by end-to-end stacking at\nsilicon studies (Fig. S18), including the AT pair closing to the terminal the terminals, forming reversible linear aggregates (Fig. 3G). Upon the\nof DNA segment (AT-DNA; I; Fig. 3F), the CG pair near the other ter\u00ad insertion of Ru1, two additional interaction patterns are observed. The\nminal of DNA segment (CGd-DNA; II) and the CG pair in the middle of first interaction pattern is a T-shaped pattern arising from the interac\u00ad\nthe DNA (CGm-DNA; III). Furthermore, three Ru1 molecules inserted tion between the tail group of one DNA segment inserted by Ru1\ninto the DNA segments at the positions of I, II, and III were constructed molecule and the terminal base of the other DNA segment (Fig. 3H). The\nand named AT-CGd-CGm-DNA. A system with only 15 copies of DNA second interaction pattern is the inter-molecular interactions of Ru1,\nsegments was also modeled for comparison (Fig. 3G). especially the interactions between the P(Ph)3 groups (Fig. S24). The T-\n The initial MD conformation consists of 15 copies of DNA segments shaped pattern is well maintained during the MD simulations, while the\nseparated by 40 \u00c5, and the DNA aggregation process is indicated by the later interactions are dynamic and interchangeable due to the flexibility\ndecrease in the total number of clusters (Figs. S19\u2013S22). Due to the finite of the long alkyl chain.\nsize of our MD simulation system with periodic boundary conditions\n(PBC), the aggregation process occurs during the first 400 ns in all of our\nMD simulation systems. The total number of clusters begins to decrease\nfrom 15 and reaches equilibrium after 400 ns. Furthermore, the\n\n\n 4\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\n\n\nFig. 3. Binding properties of Ru1 with DNA. (A and B) UV\u2013vis titration and fluorescence titration of Ru1 (30.0 \u03bcM) with CT-DNA in Tris-HCl buffer (pH 7.4, with 1 %\nDMSO). The arrows show the changes in the absorbance upon addition of CT-DNA. (C) CD spectroscopy of dsDNA in Tris-HCl buffer (pH 7.4, 100 mM NaCl) in the\npresence of different concentrations Ru1. (D and E) Agarose gel electrophoresis of pBR322 plasmid (4361 bp; 10 ng uL\u2212 1) in the absence and presence of light (12 J\ncm\u2212 2, i.e. 20 mW cm\u2212 2 for 10 min). DNA was incubated different concentrations of Ru1. Form I/II/III: supercoiled/nicked/linear DNA. (F) MD-simulated binding\nstructure of Ru1 with AT-DNA. The mainchain of DNA is shown in green, the upper and lower layers of \u03b2-carboline are labelled in purple. Ru1 is shown in yellow\nsphere-sticks. (G) Snapshots of DNA aggregation in AT-CGd-CGm-DNA model (left panel) and DNA-only model (right panel), DNA segments located in the periodic\nimages are shown in white surface representations. (H) The stabilization of T-shaped binding arises from the p-\u03c0 interaction between the long alkyl chain and the\nterminal base pair of DNA.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n\n2.4. Ru1 NPs can activate cGAS-STING pathway by photo-damaging (DCFH-DA) staining and flow cytometry (Fig. 4A). At a concentration of\nmtDNA 1.5 \u03bcM, Ru1-mediated PDT results in an approximately 20-fold increase\n in cellular ROS levels. After Ru1 treatment in the presence light, most\n Ru1-mediated PDT causes a dose-dependent increase in the cellular cells show depolarized mitochondrial with lost mitochondrial mem\u00ad\nROS levels as measured by 2\u2032,7\u2032-dichlorodihydrofluorescein diacetate brane potential (MMP), as evidenced by 5,5\u2032,6,6\u2032-tetrachloro-1,1\u2032,3,3\u2032-\n\n\n\n\nFig. 4. (A) Flow cytometry measurement of cellular ROS in MDA-MB-231 cells treated with Ru1 at the indicated concentrations for 12 h in the presence of light (425\nnm; 12 J cm\u2212 2). (B) Impact of Ru1 on MMP measured by JC-1 staining and flow cytometry. \u03bbex = 488 nm; \u03bbem = 530 \u00b1 30 nm (Green)/590 \u00b1 30 nm (Red). MDA-MB-\n231 cells were treated with Ru1 at the indicated concentrations for 12 h in the presence of light (425 nm, 12 J cm\u2212 2). (C) Confocal images of Ru1-treated MDA-MB\n231 cells stained with PicoGreen and MTDR for 15 min. Cells were treated with Ru1 at the indicated concentrations for 12 h in the presence of light (425 nm, 12 J\ncm\u2212 2). (D) TEM images of MDA-MB-231 cells treated with Ru1 (0.5 \u03bcM) for 12 h and irradiated with a 425 nm laser (12 J cm\u2212 2). (E) Immunofluorescence assay of 8-\nOG in MDA-MB-231 cells treated with Ru1 at the indicated concentrations for 6 h in the presence of light (425 nm, 12 J cm\u2212 2). 8-OG: \u03bbex = 488 nm; \u03bbem = 518 \u00b1 20\nnm. DAPI: \u03bbex = 405 nm; \u03bbem = 461 \u00b1 20 nm. (F) Impact of Ru1-mediated PDT on the transcription levels of mitochondria-encoded genes analyzed by RT-qPCR.\nMDA-MB 231 cells were treated with Ru1 (1 \u03bcM) for 12 h, irradiated with 425 nm (12 J cm\u2212 2) light array. (G) Protein expression levels of cGAS-STING\nsignaling pathway related proteins in MDA-MB-231 cells after treatment with different concentration of Ru1 in the presence of light (425 nm, 12 J cm\u2212 2).(For\ninterpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)\n\n 5\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\ntetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining (Fig. 4B). cGAS-STING pathway by photo-damaging mtDNA.\nAbout 32 % of cells lost MMP after they are treated with Ru1 at 2 \u03bcM\nupon irradiation. Morphological observation also shows that mito\u00ad 2.5. Impact of Ru1 treatment on transcriptome\nchondria change from a filament to dot-like pattern in cells subjected to\nRu1-mediated PDT treatment (Fig. 4C). Meanwhile, Picogreen reveals We further investigated the impact of Ru1 on transcriptome by RNA-\nthe good colocalization with MitoTracker Deep Red (MTDR) in the sequencing. Compared with the control group, 215 differentially\ncontrol cells with a high Pearson\u2019s correlation coefficient (PCC: 0.72, expressed genes (DEGs; |fold change (FC)| \u2265 2; false discovery rate\nFig. S25A). After incubated with Ru1 for 12 h and irradiated at 425 nm (FDR) \u2264 0.05) were detected in cells treated with Ru1 combined with\nlaser for 5 or 15 min, the localization of Picogreen was gradually sepa\u00ad light, of which 80 were significantly upregulated and 135 were signifi\u00ad\nrated from MTDR (PCC: 0.35 and 0.40, Figs. S25B and S25C), indicating cantly downregulated (Fig. 5A). Cluster analysis and Heatmap displays\nthat mtDNA was released from mitochondria. Correspondingly, Trans\u00ad that the differentially expressed genes induced by Ru1-treated group\nmission electron microscope (TEM) observation shows that mitochon\u00ad show significant differences in expression patterns compared with the\ndria in the treated cells seem to fuse together (Fig. 4D). These results control group, indicating repeatability of the data (Fig. 5B).\nshow that Ru1-mediated PDT can significantly elevate ROS levels and Gene Ontology (GO) enrichment analysis shows that Ru1-mediated\naffect mitochondrial functions. PDT mainly affects regulation of cell communication, molecular func\u00ad\n Immunostaining of 8-oxoguanine (8-OG) staining shows that oxida\u00ad tion and binding (Figs. S27\u2013S29). Kyoto Encyclopedia of Genes and\ntive damage of DNA caused by Ru1-mediated PDT mainly occurs in Genomes (KEGG) enrichment analysis shows that Ru1 mainly affects the\nmitochondria, rather than in the nuclei (Fig. 4E). Reverse transcription PI3K-AKT signaling pathway, Interleukin 17 (IL-17) signaling pathway,\nquantitative polymerase chain reaction (RT-qPCR) also shows that the tumor necrosis factor (TNF) signaling pathway and hypoxia inducible\ntranscription of 13 genes encoded by mtDNA is significantly down- factor 1\u03b1 (HIF-1\u03b1) signaling pathway (Fig. 5C). Gene set enrichment\nregulated in cells subjected to Ru1-mediated PDT (Fig. 4F). analysis (GSEA) shows that in cells treated with Ru1-treated group,\n Because mtDNA damage may lead to the release of cytoplasmic DNA, pathways related to translation, cellular respiration, mitochondrial\nthus activating the cGAS-STING signal pathway [58], we first detected electron transport, nicotinamide adenine dinucleotide (NADH) to ubi\u00ad\nthe existence of dsDNA in the cytoplasm. Picogreen staining shows that quinone and oxidation-reduction process are upregulated (Fig. 5D). The\nlarge and bright mitochondrial nucleoids form in the cytoplasm after result is consistent with the fact that Ru1 can localized in mitochondria\nRu1 treatment in combination with light (Fig. 4C), which is one of the to impact the material and energy metabolism of tumor cells, and it can\nmarkers of mtDNA damage [50]. The expression of the marker of DNA also modulate the immune function of tumor cells.\ndamage response, \u03b3H2AX is increased [59]. Accordingly, Ru1-mediated\nPDT increases the expression of cGAS and phospho-STING (pSTING;\nFig. 4G and S26). These results indicate that Ru1 can activate the\n\n\n\n\nFig. 5. Impact of Ru1 on transcriptome. (A) Volcano plots show the differentially expressed genes in Ru1-treated (0.5 \u03bcM, 12 h) MDA-MB-231 cells in the presence of\n425 nm laser (12 J cm\u2212 2). (B) Cluster analysis and Heatmap displays the overview of the differentially expressed genes. (C) KEGG enrichment analysis of the DEGs\nafter Ru1-mediated PDT treatment. (D) GSEA reveals negative and positive enrichment of genes altered in various cellular processes. NES: normalized enrich\u00ad\nment score.\n\n 6\n\fY.-Y. Ling et al. European Journal of Medicinal Chemistry 275 (2024) 116638\n\n\n2.6. Ru1 activate cancer immune responses in vivo days (Fig. 6A).\n Compared with the control, the growth of both primary tumors and\n In order to validate the phototherapeutic effects of Ru1 in vivo, distant tumors (Fig. 6B and D) is significantly inhibited upon light\nvaccination experiments were performed in BALB/C female mice irradiation. At the end of treatment, the primary tumors and distant\nbearing 4T1 tumors. The bilateral subcutaneous 4T1 tumor models are tumors in the PDT treatment groups decrease by about 58 % and 84 %,\nideal cancer vaccine models for antitumor immunotherapy research respectively. No significant change in weight of mice was detected,\n[50]. 4T1 cells were subcutaneously inoculated to the BALB/C mice as which shows that Ru1 and Ru2 possess no obvious side effects (Fig. 6C).\nprimary tumors. A week later, living 4T1 cells were subcutaneously into Hematoxylin and eosin (H&E) staining of the main organs shows no\nthe contralateral as distant tumors. 2 days later, the primary tumors serious structural and pathological alternations in all the treatment\nwere intratumorally injected with Ru1 and treated with a 425 nm light groups (Fig. S30). Ru1 has a more significant antitumor effect than Ru2\narray (12 J cm\u2212 2) after 12 h. The treatment was given every 7 days. The on both proximal and distal tumors, which may be due to the capability\ntumor volume and the weight of mice were routinely monitored for 14 of Ru1 to self-assemble into NPs for more effective retention in tumors.\n\n\n\n\nFig. 6. Ru1 activate cancer immune responses in vivo. (A) Schematic diagram of in vivo photo-immunotherapy procedure. (B) Representative images of tumors at the\nend of treatment. (C) The weights of the mice during the treatment. 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