Mitochondrial Viscosity Probes: Iridium(III) Complexes Induce Apoptosis in HeLa Cells.

{"full_text": " Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n www.chembiochem.org\n\n\n Mitochondrial Viscosity Probes: Iridium(III) Complexes\n Induce Apoptosis in HeLa Cells\n Bingbing Chen\u2020,[a, c] Zhijun Liang\u2020,[a] Yao Gong\u2020,[a] Wei Wu,[a] Jiaen Huang,[a] Jiaxi Chen,*[a]\n Yanmei Wang,[a] Jun Mei,[a] Rui Chen,*[a] Zunnan Huang,[b] and Jing Sun*[a, b]\n\n Mitochondrial viscosity has emerged as a promising biomarker (ROS), which triggered a cascade of events leading to mitochon-\n for diseases such as cancer and neurodegenerative disorders, drial dysfunction. Additionally, the fluorescence lifetime of Ir1\n yet accurately measuring viscosity at the subcellular level demonstrated high sensitivity to intracellular viscosity changes,\n remains a significant challenge. In this study, we synthesized enabling real-time fluorescence lifetime imaging microscopy\n and characterized three cyclometalated iridium(III) complexes (FLIM) of cellular micro-viscosity during apoptosis. These\n (Ir1-Ir3) containing 5-fluorouracil derivatives as ligands. Among findings underscore the potential of cyclometalated Ir(III)\n these, Ir1 selectively induced apoptosis in HeLa cells by complexes for both therapeutic and diagnostic applications at\n increasing mitochondrial production of reactive oxygen species the subcellular level.\n\n\n 1. Introduction external factors, including concentration and solvent\n conditions.[14]\n Cancer represents a growing global health and economic Fluorescence lifetime, an intrinsic characteristic of fluores-\n challenge, with incidence rates projected to increase signifi- cent molecules, is highly sensitive to changes in the molecular\n cantly in the coming years. Although cisplatin remains a microenvironment. Fluorescence lifetime imaging microscopy\n cornerstone of anticancer therapy, its efficacy is often compro- (FLIM), a non-invasive and minimally destructive technique,\n mised by drug resistance and severe side effects,[1\u20133] prompting enables precise monitoring and visualization of these changes\n the search for alternative therapies involving other transition within cells,[15] offering high specificity and sensitivity while\n metals. Among these, iridium has attracted considerable remaining unaffected by common issues such as photobleach-\n attention due to its distinctive properties, including its ability to ing, excitation variability and fluorescence intensity\n form stable complexes suitable for targeted therapies. Iridium fluctuations.[16] Additionally, FLIM probes with viscosity-depend-\n complexes offer several advantages, such as tunable config- ent lifetimes can provide quantitative insights into cellular\n urations, exceptional photophysical properties, organelle-tar- states, such as inflammation and programmed cell death.\n geting capabilities and distinct antitumor mechanisms.[4,5] For Mitochondria, often referred to as the \u201cpowerhouses\u201d of the\n example, Ir(III) complexes can be designed to target specific cell, play a critical role in maintaining cellular metabolism and\n organelles through molecular design and structural homeostasis.[17] Changes in mitochondrial viscosity are closely\n modifications.[6,7] Furthermore, these complexes can detect linked to physiological processes, including metabolite diffu-\n various environmental changes, such as shifts in pH,[8,9] oxygen sion, electron transport, apoptosis, autophagy, mitosis and\n levels[10,11] and viscosity.[12,13] However, their application as protein-protein interactions.[18,19] Importantly, mitochondrial vis-\n fluorescence probes has been limited by low sensitivity to cosity has been identified as a potential biomarker for several\n diseases, including cancer,[20] Huntington\u2019s disease[21] and\n Alzheimer\u2019s disease.[22,23]\n In this study, we aimed to leverage the unique photo-\n [a] B. Chen, Z. Liang, Y. Gong, W. Wu, J. Huang, J. Chen, Y. Wang, J. Mei,\n R. Chen, J. Sun physical properties of cyclometallic Ir(III) complexes incorporat-\n The First Dongguan Affiliated Hospital, School of Pharmacy, Guangdong ing 1-(4-((1,10-phenanthrolin-5-yl)amino)butyl)-5-fluoropyrimi-\n Medical University, Dongguan 523808, China dine-2,4(1H,3H)-dione (L) as a ligand to detect mitochondrial\n Tel.: + 86-769-2289-6322 viscosity while inducing antitumor activity. Three Ir(III) com-\n E-mail: jiaxi@gdmu.edu.cn\n 415317456@qq.com plexes were synthesized, each incorporating a different com-\n sunjing03@foxmail.com pound represented as (C N) in the chemical formula [Ir-\n [b] Z. Huang, J. Sun (C N)2L]PF6: Ir1 (1-phenylisoquinoline, piq), Ir2 (7,8-\n Key Laboratory of Computer-Aided Drug Design of Dongguan City, benzoquinoline, bzq) and Ir3 (2-(2-thienyl)pyridine, thpy). Ir1\n Guangdong Medical University, Dongguan 523808, China\n specifically targeted mitochondria in HeLa cells, inducing\n [c] B. Chen\n apoptosis by generating reactive oxygen species (ROS), which\n Key Laboratory of Luminescence Analysis and Molecular Sensing, College of\n Pharmaceutical Sciences, Southwest University, Chongqing 400715, China triggered a cascade of events leading to mitochondrial\n [\u2020] Contributed equally to this work. dysfunction. Additionally, the fluorescent properties of Ir1\n Supporting information for this article is available on the WWW under enabled quantitative, real-time monitoring of changes in\n https://doi.org/10.1002/cbic.202400756 mitochondrial viscosity through FLIM. This study highlights the\n\n\n ChemBioChem 2025, 26, e202400756 (1 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n potential of cyclometallic Ir(III) complexes to induce and the glycerol concentration increased to 90 %, the T values for\n precisely monitor microenvironmental changes at the subcel- Ir1, Ir2 and Ir3 increased from 0.35 \u03bcs to 3.2 \u03bcs, 0.15 \u03bcs to\n lular level, offering valuable insights for therapeutic and 1.26 \u03bcs and 0.12 \u03bcs to 4.5 \u03bcs, respectively. A direct linear\n diagnostic applications (Figure 1). correlation was observed between the T values of Ir1-Ir3 and\n the solvent viscosity parameter (\u03b7), consistent with the range of\n viscosities reported in cellular environments.[25,26] Overall, all\n 2. Results and Discussion three complexes exhibited enhanced photophysical properties,\n with Ir1 demonstrating the most significant increase in\n 2.1. Synthesis and Characterization fluorescence intensity, prompting further studies on its physical\n properties.\n Ligand L was synthesized following our previously established To confirm the specificity of Ir1\u2019s response to viscosity, we\n method.[24] The synthesis of complexes Ir1-Ir3 is outlined in evaluated its fluorescence lifetime under various conditions.\n Scheme 1. Briefly, two equivalents of ligand L were refluxed Increasing the proportion of 1,4-dioxane in water from 25 % to\n with a chlorine-bridged Ir(III) dimer. The resulting complexes 100 % resulted in minimal changes in the T value of Ir1\n Ir1-Ir3, were characterized via elemental analysis, ESI-MS, and (Figure S9). Additionally, its fluorescence lifetime showed only\n 1\n H NMR, as shown in Figure S1\u2013S6. The UV-Vis absorption slight responses to variations in solvent type, glutathione (GSH)\n spectra of Ir1-Ir3 in phosphate-buffered saline (PBS), CH2Cl2 and content and human serum albumin (HSA) content (Figure S10).\n CH3CN exhibited pronounced absorption in the 270\u2013300 nm\n range, corresponding to spin-allowed intra-ligand electron\n transitions, and weaker absorption in the 400\u2013450 nm range, 2.3. Cytotoxicity Assay\n indicative of a metal-to-ligand electronic transition (Figure S7).\n Upon excitation at 405 nm, all complexes emitted light in PBS, The cytotoxicities of the Ir1-Ir3 were assessed against various\n CH2Cl2 and CH3CN at 298 K (Figure S8). tumor cell lines, including HeLa (human cervical carcinoma),\n HepG2 (human hepatocellular carcinoma), MCF-7 (Michigan\n Cancer Foundation-7, human breast carcinoma), Caco-2 (human\n 2.2. Viscosity-Sensitive Emission Properties colon adenocarcinoma) and KYSE-140 (human esophageal\n squamous cell carcinoma). All three modified 5-Fu Ir(III) com-\n To evaluate the viscosity-sensitive properties of complexes Ir1- plexes exhibited significantly enhanced antitumor activity in\n Ir3, we examined their fluorescence intensity and lifetime in HeLa cells compared to 5-Fu alone (Table 1). Among them, Ir1\n various solvent mixtures of methanol and glycerol. As shown in exhibited the strongest antiproliferative effect, with an IC50\n Figure 2, all three complexes displayed viscosity-dependent value of 3 \u03bcM against HeLa cells. Given Ir1\u2019s excellent photo-\n changes in both fluorescence intensity and fluorescence life- physical properties and potent antitumor activity, it was\n time (T). Notably, the fluorescence intensity of Ir1 in 80 % selected for further mechanistic studies.\n glycerol was 10.2 times higher than in methanol, while Ir2 and\n Ir3 showed 8.73-fold and 4.09-fold increases, respectively. As\n\n\n\n\n Figure 1. The structures of complexes Ir1-Ir3.\n\n\n ChemBioChem 2025, 26, e202400756 (2 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n\n\n Figure 2. Emission intensity (A) and lifetime (B) of complexes Ir1-Ir3 measured in mixtures of CH3OH and glycerol (percentage of glycerol = 0 %, 10 %, 20 %,\n 30 %, 40 %, 50 %, 60 %, 70 %, 80 and 90 %, v/v, \u03bbex = 405 nm). The arrow shows the intensity variations with an increasing proportion of glycerol.\n\n\n\n Table 1. IC50 values of tested compounds towards different cell lines.[a]\n Compounds IC50 (\u03bcM)\n HeLa HepG2 MCF-7 Caco-2 KYSE-140\n\n Ir1 3.0 \ufffd 0.1 105.6 \ufffd 4.6 9.3 \ufffd 1.3 14.6 \ufffd 1.7 6.1 \ufffd 0.2\n Ir2 5.4 \ufffd 0.1 129.1 \ufffd 5.8 34.2 \ufffd 3.1 21.8 \ufffd 2.0 14.9 \ufffd 1.9\n Ir3 6.4 \ufffd 0.3 77.3 \ufffd 3.8 53.8 \ufffd 4.2 19.6 \ufffd 5.5 19.3 \ufffd 2.5\n 5-Fu 47.9 \ufffd 1.7 42.1 \ufffd 2.3 65.0 \ufffd 2.6 11.4 \ufffd 1.0 58.6 \ufffd 3.3\n\n [a] IC50 values are drug concentrations necessary for 50 % inhibition of cell viability. Data are presented as \ufffd standard deviations obtained in at least three\n independent experiments and treatment period was 48 h.\n\n\n\n\n ChemBioChem 2025, 26, e202400756 (3 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n 2.4. Intracellular Localization totic cells following 24-hour Ir1 treatment, rising from 0.76 \ufffd\n 0.08 % at 0 \u03bcM to 59.75 \ufffd 2.33 % at 6 \u03bcM Ir1 (Figure 4B).\n Based on their excellent photophysical properties, we utilized Caspase proteins play a crucial role in the regulation of\n fluorescence microscopy to visualize the subcellular distribu- apoptosis and can be categorized into initiator caspases (e. g.,\n tions of Ir(III) complexes. Confocal microscopy images (Fig- caspase 8 and caspase 9) and executioner caspases (e. g.,\n ure S11) revealed that Ir1 accumulated in the cytoplasm after caspase 3 and caspase 7) based on their functions.[28] During\n 5 hours of treatment. Given that mechanistic studies suggest apoptotic stress, increased mitochondrial outer membrane\n small molecules like Ir1 enter cells via energy-dependent permeability allows the release of apoptotic proteins from the\n pathways, we conducted confocal microscopy on HeLa cells intermembrane space into the cytoplasm. This release sub-\n exposed to Ir1 at a low temperature (4 \u00b0C) and observed sequently activates caspases 3 and 7, initiating apoptosis.\n reduced uptake efficiency (Figure S12), confirming an energy- Therefore, elevated levels of caspase 3/7 activity are indicative\n dependent uptake mechanism. of apoptosis induction.[29,30] Following 24-hour treatment of\n Previous studies have reported that Ir(III) complexes localize HeLa cells with Ir1, we observed a dose-dependent increase in\n to specific organelles, such as mitochondria or lysosomes.[27,28] caspase 3/7 activity, with the highest concentration group\n To further investigate Ir1 localization in HeLa cells, we used showing a 2.82-fold increase compared to the control group\n MitoTracker Red (MTR) and LysoTracker Red (LTR) dyes. After a (Figure 4C). These findings collectively suggest that Ir1 induces\n 5-hour incubation, Ir1 showed substantial colocalization with apoptosis in HeLa cells through a caspase-dependent mito-\n MTR, as indicated by a Pearson\u2019s correlation coefficient (PCC) of chondrial pathway.\n 0.94, which was significantly higher than its colocalization with\n LTR (PCC = 0.39) (Figure 3), demonstrating the selective mito-\n chondrial localization of Ir1. 2.6. Intracellular ROS Level\n\n Research has demonstrated that elevated ROS levels can\n 2.5. Apoptosis Induction damage tumor cells and induce apoptosis.[31,32] To assess this\n effect, HeLa cells were incubated with Ir1 at concentrations of\n Given Ir1\u2019s superior antitumor activity compared to 5-Fu, we 2, 4 and 6 \u03bcM for 12 hours, and intracellular ROS levels were\n further investigated its mechanism of action. Apoptosis is measured using H2DCF-DA staining. Flow cytometry results\n characterized by distinct morphological changes, including cell (Figure 5A) showed that ROS levels in the highest concentration\n shrinkage, mitochondrial swelling, formation of apoptotic group were 5.7 times greater than those in the control group.\n bodies and nuclear fragmentation.[27] Confocal microscopy data Confocal microscopy confirmed a concentration-dependent\n (Figure 4A) showed that treatment of HeLa cells with Ir1 led to increase in intracellular ROS levels (Figure 5B), consistent with\n dose-dependent nuclear damage, with significant nuclear the flow cytometry findings. The fluorescence intensity of the\n fragmentation occurring at higher Ir1 concentrations. Consis- DCF probe within the cells also exhibited a concentration-\n tent with these findings, Annexin V-FITC staining revealed a dependent increase, suggesting that Ir1 can induce a significant\n concentration-dependent increase in the proportion of apop- rise in ROS production in HeLa cells.\n\n\n\n\n Figure 3. Confocal microscopy images of HeLa cells co-labeled with Ir1 (2 \u03bcM, 5 h), MTR (100 nM, 0.5 h) and LTR (100 nM, 0.5 h). The excitation of Ir1 was\n 405 nm, whereas MTR and LTR were 552 nm.\n\n\n ChemBioChem 2025, 26, e202400756 (4 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n\n\n Figure 4. (A) Confocal microscopic images of HeLa cells incubated with Ir1 (2, 4, 6 \u03bcM, 24 h) and stained with Hoechst 33342 (5 \u03bcg/mL, 30 min). \u03bbex = 405 nm;\n \u03bbem = 406 \ufffd 20 nm. (B) Flow-cytometric quantification of Annexin V-FITC (FITC: fluoresceine isothiocyanate) labeled HeLa cells treated with Ir1 at different\n concentrations for 24 h. (C) Activation of caspase 3/7. HeLa cells were exposed to Ir1 at different concentrations for 24 h. (*p < 0.05, **p < 0.01, n = 9)\n\n\n\n\n Figure 5. Effects of Ir1 on ROS generation. HeLa cells were treated with Ir1 at different concentrations for 24 h, and stained by DCFH-DA. Samples were\n detected by flow cytometryy (A) and confocal microscop (B).\n\n\n\n\n 2.7. Cell-Cycle Arrest Table S1). These findings indicate that Ir1 induces apoptosis by\n targeting actively replicating DNA and arresting cells in the S\n Iridium-based transition metal complexes and the conventional phase, reinforcing DNA damage as a key mechanism underlying\n anti-cancer drug 5-Fu typically induce apoptosis by damaging its antitumor activity.\n DNA,[33,34] prompting us to use flow cytometry to assess DNA\n content in Ir1-treated HeLa cells. After HeLa cells were treated\n with 2, 4 and 6 \u03bcM Ir1 for 24 hours, a significant proportion of 2.8. Mitochondrial Dysfunction\n cells were arrested in the S phase of the cell cycle (Control:\n 46.04 \ufffd 1.04 %; 6 \u03bcM Ir1: 55.46 \ufffd 1.41 %), accompanied by a Mitochondria are essential for ATP production via the tricarbox-\n marked reduction in the percentage of cells in the G1/G0 phase ylic acid cycle;[35,36] therefore, disruption of mitochondrial\n (Control: 34.05 \ufffd 0.39 %; 6 \u03bcM Ir1: 17.28 \ufffd 1.45 %) (Figure 6 and function reduces ATP production, impairing cellular processes\n\n\n ChemBioChem 2025, 26, e202400756 (5 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n\n\n Figure 6. Effects of complexes Ir1 on cycle distribution of HeLa cells. Cells were treated with Ir1 at different concentrations for 24 h and analyzed by flow\n cytometry after PI staining (*p < 0.05, **p < 0.01, n = 9)\n\n\n\n\n such as mitosis, apoptosis and autophagy.[37] Here we assessed fluorescence lifetime was observed, showcasing its ability to\n HeLa cell viability after 24-hour Ir1 treatment using the track real-time changes in mitochondrial viscosity (Figure 8).\n CellTiter-Glo\u00ae Luminescent Cell Viability Assay. As shown in The average fluorescence lifetime increased from 1748.88 ns to\n Figure 7A, cell viability decreased to 52.95 % at the highest 2345.34 ns, corresponding to a rise in mitochondrial viscosity\n concentration (6 \u03bcM Ir1) compared to the control (0 \u03bcM Ir1). from 65.69 cP to 143.72 cP. These results align with previously\n Mitochondrial physiological status was further evaluated reported data[39] and suggest a correlation between mitochon-\n using JC-1 dye, which shifts from red to green fluorescence in drial viscosity and physiological status.\n response to mitochondrial damage or depolarization.[38] Ir1\n treatment led to a concentration-dependent decrease in red\n fluorescence and a corresponding increase in green 3. Conclusions\n fluorescence, indicating mitochondrial depolarization and dam-\n age in HeLa cells (Figure 7B). In this study, we synthesized three iridium(III) complexes (Ir1-\n Ir3) containing 5-Fu, all of which demonstrated excellent\n photophysical properties. Among these, Ir1 exhibited a specific\n 2.9. Tracking of Mitochondrial Viscosity via FLIM response to environmental viscosity and effectively accumu-\n lated in mitochondria, enabling precise labeling of these\n The fluorescence lifetime of Ir1 is linearly related to environ- organelles. Furthermore, Ir1 retained the properties of 5-Fu by\n mental viscosity. Using FLIM, we monitored dynamic changes in impairing cellular DNA function, blocking cell cycle progression\n mitochondrial viscosity within HeLa cells following Ir1 exposure. and inducing apoptosis through caspase activation. Addition-\n After 4 hours of treatment, a measurable increase in Ir1 ally, Ir1 significantly disrupted mitochondrial energy and\n\n\n ChemBioChem 2025, 26, e202400756 (6 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License\n Research Article\nChemBioChem doi.org/10.1002/cbic.202400756\n\n\n\n\n Figure 7. Effects of Ir1 on mitochondrial integrity. (A) Intracellular ATP levels in Hela cells treated with Ir1 at different concentrations for 24 h. (B) Cells were\n treated with Ir1 at different concentrations for 12 h and analyzed by flow cytometry after JC-1 staining. (**p < 0.01, n = 9)\n\n\n\n\n Figure 8. Mitochondrial viscosity in Ir1-treated HeLa cells detected by FLIM. HeLa cells were treated with Ir1 (10 \u03bcM) and imaging at 1, 2, 3, and 4 h.\n (\u03bbex = 405 nm, \u03bbem = 600 \ufffd 20 nm)\n\n\n\n\n metabolic states, leading to increased production of mitochon- Province (2024ZDZX2070), Medical Industry Innovation Project\n drial ROS. Due to its highly sensitive fluorescence lifetime of Guangdong Medical University (4SG22305P), and Medical\n response to mitochondrial viscosity, Ir1 is an effective tool for Scientific Research Foundation of Guangdong Province\n monitoring mitochondrial viscosity changes using FLIM. Our (B2023240).\n findings provide new insights into the development of small\n molecule-based anticancer drugs that not only induce ther-\n apeutic effects but also enable the real-time monitoring of Conflict of Interests\n microenvironmental changes at the subcellular level.\n The authors declare no competing financial interests.\n\n Acknowledgements\n Data Availability Statement\n This work was funded by the Discipline Construction Project of\n Guangdong Medical University (4SG23004G), Key Scientific The data that support the findings of this study are available in\n Research Projects of Colleges and Universities in Guangdong the supplementary material of this article.\n\n\n ChemBioChem 2025, 26, e202400756 (7 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f 14397633, 2025, 3, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202400756 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. 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Version of record online: November 23, 2024\n\n\n\n\n ChemBioChem 2025, 26, e202400756 (8 of 8) \u00a9 2024 Wiley-VCH GmbH\n\f", "pages_extracted": 8, "text_length": 38675}