Unraveling Mechanism and Enhancing Selectivity of a Ru^II-bis-bipyridyl-morphocumin Complex with RAFT-Generated Glycopolymer Exploiting Warburg Effect in Cancer.

{"full_text": " Research Article\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n www.chemeurj.org\n\n\n Unraveling Mechanism and Enhancing Selectivity of a RuII-\n bis-bipyridyl-morphocumin Complex with RAFT-Generated\n Glycopolymer Exploiting Warburg Effect in Cancer\n Souryadip Roy,[a] Soumya Paul,[a] Sujato Mukherjee,[a] Priyadarsi De,[a] and\n Arindam Mukherjee*[a]\n\n The Warburg effect, which generates increased demand of morphocumin release through a synergistic domino effect.\n glucose in cancer cells is a relatively underexplored phenomen- Comparative studies reveal that 2 outperforms its curcumin\n on in existing commercial drugs to enhance uptake in cancer Ru(II) complex (1) analog in solution stability, organelle\n cells. Here, we present a chemotherapeutic strategy employing specificity, and cellular mechanisms. Both 1 and 2 exhibit\n a Ru(II)-bis-bipyridyl-morphocumin complex (2) encapsulated in phototherapeutic effects under low-intensity visible light, but\n a self-assembling glucose-functionalized copolymer P(G-EMA- their chemotoxicity significantly increases with incubation time\n co-MMA) (where G = glucose; MMA = methyl methacrylate; in the dark, highlighting the superior chemotherapeutic efficacy\n EMA = ethyl methacrylate), designed to exploit this effect for of the O,O-coordinating Ru(II) ternary polypyridyl complexes.\n enhanced selectivity in cancer treatment. The P(G-EMA-co- Complex 2 induces apoptosis via the intrinsic pathway and\n MMA) polymer, synthesized via reversible-addition fragmenta- shows a 9-fold increase in selectivity for pancreatic cancer cells\n tion chain transfer (RAFT) polymerization, has a number average (MIA PaCa-2) over non-cancerous HEK293 cells when encapsu-\n molecular weight (Mn,NMR) of 8000 g/mol. Complex 2, stable in lated in the glucose-conjugated polymer (DP@2). Glucose\n aqueous media, selectively releases a cytotoxic, lysosome- deprivation in the culture medium further enhances drug\n targeting compound, morphocumin, in the presence of excess efficacy by an additional 5-fold. This work underscores the\n hydrogen peroxide (H2O2), a reactive oxygen species (ROS) potential of glucose-functionalized polymers and ROS-respon-\n prevalent in tumor microenvironments. Additionally, complex 2 sive Ru(II) complexes in targeted cancer therapy.\n promotes ROS accumulation, which may further enhance\n\n\n Introduction loss due to thiol-based sequestration, researchers are drawn to\n the potential of Ru polypyridyl complexes. These complexes\n Over decades, researchers have delved into the complexities of offer straightforward synthesis, exceptional stability, a broad\n cancer, progressively gaining deeper insights. Among the array range of photophysical and photochemical properties, and\n of drugs utilized in cancer chemotherapy, platinum complexes significant activity in biological systems.[4,5] [Ru(bpy)3]Cl2 was the\n have exhibited significant success with dominance, albeit first reported ruthenium polypyridyl complex by Burstall in\n accompanied by severe side effects, a common drawback 1936[6] but gained prominence in the early 1980s for capability\n shared among most anti-cancer medications.[1] Notably, the soft of water splitting through the \u2019ruthenium blue dimer\u2019 [(bpy)2Ru-\n nature of Pt(II) is exploited by cancer cells, rendering the drugs (H2O)(\u03bc-O)(H2O)Ru(bpy)2]4 +.[7] In the past decade, Ru polypyridyl\n less effective by coordinating with thiols from various small complexes have emerged as promising metal-based anticancer\n molecules and proteins abundant in cancer cells (e. g., gluta- agents, particularly in photodynamic therapy (PDT).[8\u201310] This\n thione, ATP7B, MMP, thioredoxin, etc.). In the quest for non- interest was further fueled by the entry of the first Ru(II)-based\n Pt(II) anti-cancer agents, Ru(II) presents a promising alternative polypyridine, TLD-1433, into phase II clinical trials for non-\n due to its lower softness stemming from a greater charge/ muscle invasive bladder cancer.[10,11] Various research groups\n radius ratio rendering subtle preferential differences in coordi- have highlighted the potential of these complexes in both PDT\n nation environment, which impart suitable biological proper- and photoactivated chemotherapy (PACT).[12,13] PACT, also\n ties, thereby advocating its potential candidacy in cancer known as \u2019photocaged complexes\u2019, involves molecular changes\n therapeutics.[2,3] To exploit kinetic inertness and mitigate dosage upon photoirradiation, offering several advantages over tradi-\n tional PDT. Ru polypyridyl complexes with bioactive ligands are\n particularly advantageous for PACT, as both the aquated Ru\n [a] S. Roy, S. Paul, S. Mukherjee, P. De, A. Mukherjee polypyridyl and the photo-dissociated ligand can synergistically\n Centre for Advanced Functional Materials, Department of Chemical target the tumor microenvironment upon photoirradiation.[14,15]\n Sciences, Indian Institute of Science Education and Research Kolkata, Nadia,\n Natural products used in food are in general well tolerant to\n West Bengal, Mohanpur 741246, India\n E-mail: a.mukherjee@iiserkol.ac.in humans and have consistently held a role in drug design as\n Supporting information for this article is available on the WWW under bioactive ligands. Curcumin, one of the most investigated\n https://doi.org/10.1002/chem.202403695 natural products is a food additive that may be taken up to\n\n\n Chem. Eur. J. 2025, 31, e202403695 (1 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n 3 mg/kg body weight per day.[16] The dose tolerance limit of approach enhances the therapeutic efficacy of the potential\n curcumin and its derivatives is influenced by the toxic products chemotherapeutic agents by improving their solubility, bio-\n formed due to its physiological instability.[17,18] Numerous compatibility, biodistribution, and circulation time within the\n studies have documented the targeted discharge of curcumin body.[35] Additionally, the polymeric nanocarriers serve to\n within cancer cells, yet the challenge persists due to curcumin\u2019s reduce the biodegradation of the drugs before reaching the\n instability in physiological environments. Consequently, to targeted site of action.\n achieve selective release of a curcumin derivative, it is Typically, cancer cells exhibit higher glucose uptake and\n imperative for it to remain stable within the tumor micro- metabolism because of their rapid growth and metastatic\n environment. Morphocumin is designed to be physiologically nature. Therefore, various human cancer cells including breast,\n stable, better soluble, lysosome targeting photoactive curcumin pancreatic, ovarian, lung, and cervical cancer cells possess\n analogue with pronounced phototherapeutic properties.[19] overexpression of glucose transporters (GLUTs).[36] Thus, target-\n Hence, we chose Morphocumin as the bioactive ligand for this ing GLUTs using glucose-based polymeric nanoparticles, suit-\n work. able for encapsulating drugs, to facilitate endocytosis within\n Apart from external photo stimuli, several other factors are cancer cells is a suitable approach for drug delivery in cancer\n the foundation of ligand ejection that generally differentiates therapy.[31] Glycopolymers consist of a synthetic polymer back-\n cancer cells from normal cells i. e., pH variation, reactive oxygen bone adorned with sugar moieties, and they have garnered\n species (ROS) overexpression, redox activity and enzyme significant attention because of the high specificity of carbohy-\n overexpression.[20] An elevated amount of ROS in hypoxic drates for sugar-binding proteins that are overexpressed on\n tumors is a defense mechanism for survival and can be cancer cell surfaces. This characteristic enables the targeting\n modulated as a therapeutic advantage. The most frequently and delivery of drugs to cancer cells through receptor-mediated\n elevated ROS include hydroperoxide (H\u022e ), superoxide (O2 ), *\n endocytosis.[37,38]\n and hydroxyl radical (\u022eH).[21] Apart from the above, hydrogen Herein, we report Ru bis-bipyridyl complexes of curcumin\n peroxide (H2O2), is a potent compound capable of generating and morphocumin differentiating their chemotherapeutic vs.\n ROS, with chemical stability needed to set up notable steady- photodynamic behavior, dependence of the activity on the O,O-\n state concentrations in vivo. Large amounts of H2O2 are coordinating curcumin derivatives coordinating to the Ru bis-\n reportedly produced in vitro without exogenous stimulation in bipyridyl precursor. Among the two complexes of the formula-\n several human carcinoma cell lines, including malignant tion [Ru(bpy)2(curcumin)]PF6 (1) and [Ru-\n melanoma, colon carcinoma, pancreatic carcinoma, neuroblas- (bpy)2(morphocumin)]PF6 (2), complex 2 display better aque-\n toma, breast carcinoma, and ovarian carcinoma.[22,23] H2O2 freely ous-stability and releases the stable lysosome targeting\n passes through membranes and can reach any cellular morphocumin in the presence of excess H2O2, a viable\n compartment.[24] Apparently human tumor cell lines constitu- therapeutic target to enhance cytotoxicity towards ROS rich\n tively generate H2O2 at rates (up to 0.5 nmol/104 cells/h) that cancer cells. The altered metabolism of neoplastic cells with\n cumulatively amount at 4 h to the amount of H2O2 produced by accelerated glycolysis, termed as \u2018Warburg effect\u2019[39] leading to\n phorbol ester-triggered neutrophils.[22] This excess availability of overexpression of GLUTs was further exploited by delivering 2\n H2O2 in cancer cells over normal cells allows the exploitation of using glucose functionalized polymeric nanoparticles\n H2O2 as a potential therapeutic target. Thus, in recent years, (GFPNs)[40,41] showcasing enhanced selectivity towards cancer\n there have been reports of utilizing H2O2 stimulation to release cells (Figure 1).\n a drug attached to a prodrug.[25,26]\n Small molecule metal complexes may often encounter\n challenges associated with poor solubility and stability in the Results and Discussion\n biological milieu, and exhibit undesired side effects resulting\n from non-specific uptake in the body.[27] Consequently, their Synthesis and Characterization of 1 and 2\n therapeutic efficacy is often compromised. To address the\n challenges of solubility, stability, and cellular uptake associated To capitalize the precedence of Ru polypyridyl complexes over\n with small molecule complexes, especially Ru(II) in particular, arene analogs, viz. strong visible light absorption, greater ROS\n researchers are developing advanced drug delivery systems generation efficiency, enhanced photophysical properties,\n aimed to improve the activity and selectivity of the stability and biocompatibility we designed simple Ru(II) bis-\n compounds.[28\u201330] In contrast to traditional systemic treatment bipyridyl complexes of O,O-coordinating ligands.[42] Two Ru\n approaches that rely on using small molecule as drugs, complexes (1 and 2) were synthesized in good yields (78 % and\n encapsulating them within nanoscale delivery vehicle can 69 % respectively) by reacting the corresponding O,O-coordinat-\n significantly enhance their stability and reduce systemic toxicity. ing ligands curcumin and morphocumin with equimolar Ru-\n This is achieved by preventing early degradation and non- (bpy)2Cl2 and 1.5 equivalent triethylamine in a 1 : 1 ethanol-\n specific interactions with healthy tissues, enhancing drug water mixture following reported protocol[43] as depicted in\n solubility, and allowing for longer circulation times in the Scheme 1. COMMENT: This scheme 1 needs to be replaced with\n bloodstream.[31] Therefore, encapsulating drugs within natural uploaded Scheme 1 as charges on PF6 were in wrong position\n or synthetic polymeric nanoparticles that impart stability and and one Cl coordinated to Ru(II) became HCl which is rectified\n selectivity are emerging as a promising strategy.[29,32\u201334] This in the uploaded scheme 1 Complex 1 was previously reported\n\n\n Chem. Eur. J. 2025, 31, e202403695 (2 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n\n\n Figure 1. Chemical structures of the previously studied Ru(II) p-cymene-morphocumin complex (2) and the Ru(II) bis-bipyridyl complexes of morphocumin\n presented in this work along with a schematic representation of the 2 encapsulated glucose functionalized polymers generating nanoparticle formulations\n and a probable mechanistic representation of the cellular mechanism of action.\n\n\n\n\n analysis. 2 in acetonitrile showed a structured spectrum in UV-\n Vis spectroscopy with absorption maxima at 295 and 398 nm\n followed by shoulders at 416 and 518 nm, which got relatively\n unstructured in water and PBS (Table 1 and Figure S6). Both the\n complexes exhibit green fluorescence with emission maxima of\n 508 (for 1) and 516 nm (for 2) with a low quantum yield of\n 0.002 and 0.0045 respectively in acetonitrile (Table 1 and\n Figure S5\u2013S8).\n\n Scheme 1. Schematic representation of the synthesis of 1 and 2 from\n curcumin and morphocumin.\n Solution Stability\n\n Ru polypyridyl complexes are mostly kinetically inert and stable\n and we used it as a standard.[44] These complexes were under physiological conditions.[5,9] The stability of both com-\n characterized by standard analytical techniques including NMR, plexes in solution was evaluated using reverse-phase high-\n ESI-HRMS, FT-IR, UV-Vis, and fluorescence spectroscopy (Fig- performance liquid chromatography (RP-HPLC) in a 10 mM\n ure S1\u2013S8). The bulk purity was ascertained by elemental phosphate buffer with a pH of 7.4, containing 4 mM NaCl\n\n\n Table 1. Photophysical properties of 1 and 2 with their corresponding ligands in acetonitrile.\n \u03bbabs (nm) \u025b (dm3mol 1 cm 1) \u03bbem (\u03bbabs) (nm) Stokes shift (nm) \u03a6f\n\n 1 296, 399, 416, 515 507(416) 91 0.002\n 2 295, 400, 416, 515 55680(295), 47050(400), 44070(416), 13230 (515) 518(416) 102 0.0045\n Curcumin 418 538(418) 120 0.075\n Morphocumin 420 57020 507(420) 87 0.081\n\n\n\n Chem. Eur. J. 2025, 31, e202403695 (3 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n (intracellular salt concentration), The HPLC analysis revealed demonstrated that variations in drug incubation time and cell\n that when dissolved in a DMSO : buffer mixture (4 : 6 v/v) at recovery time after drug removal upon light/dark treatment\n pH 7.4, a 400 \u03bcM stock solution of each complex remained lead to differences in IC50 values, evident from the photo-\n stable for up to 48 h. Complex 1 eluted at 17.8 minutes and therapeutic index (PI) shown in the comparison table (Table 2)\n complex 2 at 12.3 minutes, with no new peaks detected during (Figures S12\u2013S17). The 24 h growth post cell seeding, followed\n the 48 h period (Figures S9 and S10). This indicates that, in the by 4 h drug treatment, then washing away excess drug\n absence of an external reagent to react with Ru(II) or the followed by 1 h dark treatment/light irradiation (\u03bb = 400\u2013\n curcumin scaffold, both complexes are stable at physiological 800 nm, 2.7 mW cm 2, total 10 J cm 2) and finally 19 h incuba-\n pH of 7.4. Additionally, the aqueous solubility and stability of tion in the dark for recovery and growth (protocol 1) gave the\n complex 2 under physiological conditions were further exam- best selectivity of light vs. dark toxicity with a PI value of 3.2\u2013\n ined using UV-Vis stability kinetics with a reduced DMSO 9.6 for 2. The increase of incubation time in dark post external\n concentration. A 50 \u03bcM solution of complex 2 in phosphate drug removal and irradiation gave an extensive downfall in\n buffer at pH 7.4 containing 5 % DMSO was monitored over 24 h terms of PI though the IC50 enhanced, as evident from protocol\n via UV-Vis spectroscopy (Figure S11), confirming that 2 is stable 2 (24 h growth post cell seeding was followed by 4 h drug\n in aqueous solution. treatment, then washing away excess drug followed by 1 h light\n irradiation/dark treatment and finally 44 h incubation in the\n dark for recovery and growth). The PI became the least when\n Cytotoxicity: Light vs. Dark drug incubation time was further enhanced keeping the\n recovery time post drug removal almost same (protocol 3). The\n Over the past two decades, Ru polypyridyl complexes have observed low PI and the trend in cytotoxicity under both dark\n gained recognition primarily in the field of photodynamic and light conditions suggested that the complexes remain\n therapy.[9] Research indicates that N,N-coordinated Ru polypyr- inside the cells and exert toxicity even without light exposure.\n idyls exhibit photodynamic properties, while O,O-coordinated The low differences in PI suggested that we may conduct\n ones are generally more suitable for chemotherapy rather than further studies by using the chemotherapeutic cytotoxicity\n phototherapy due to their high dark toxicity, leading to a poor protocol (protocol 4, Table 2), exhibiting nanomolar IC50 con-\n phototherapeutic index.[43] The present study focuses on a centration (Tables 2\u20133, Figure 2 and Figures S18\u2013S19). However,\n stable curcumin derivative, which is a photoactive chromophore the phototherapeutic activity does not allow the complexes to\n capable of generating ROS when exposed to light. Conse- be in the medium for 72 h unlike protocol 4 in Table 2, rather\n quently, we examined the light vs. dark cytotoxicity of O,O- the complexes stay only for 24 h maximum in the medium and\n coordinated curcumin and morphocumin-based Ru-bis-bipyrid- then the medium is washed out and replaced with fresh\n yl complexes. We employed three different protocols, well- medium without any drug. So, we kept the drug incubation\n documented in the literature, to assess phototoxicity time 24 h (Protocol 5, Supporting information) to evaluate the\n (Table 2).[19,45] This approach provided evidence of the protocol actual potency of the chemotherapeutic agents. 1 and 2, their\n that more accurately represented phototherapeutic vs. chemo- respective ligands, and CDDP as a standard were studied in the\n therapeutic activity index of 1 and 2 (Table 2). human embryonic kidney HEK293 and pancreatic adenocarcino-\n It is worth noting that all phototherapeutic experiments\n conducted utilized low light fluence (10 J/cm2) since low fluence\n rates enhances the body\u2019s ability to fight tumors while also\n mitigating negative side effects.[46] Apparently it is found that\n the damage caused by lower PDT fluence rates tends to be\n lasting,[47] whereas higher irradiances can exhaust the oxygen\n supply in tumor tissues, resulting in less effective treatment.[46,48]\n Another advantage of using low-fluence irradiation is the\n diminished occurrence of photobleaching in photosensitizers\n during PDT treatment.[49] Thus, PDT administered at lower\n irradiances might prove more effective in preclinical trials. To\n measure chemotherapeutic activity, a standard and widely used\n protocol was followed.[50] The target cancer cells were represen-\n tative of triple-negative breast carcinoma (MDA-MB-231) and\n human pancreatic carcinomas (MIA PaCa-2 and PANC-1) which\n are known to be difficult to cure. Data revealed that the\n morphocumin based 2 has a poor phototherapeutic index (PI >\n 3). 2 was found to be more toxic in the dark compared to its\n Ruthenium-p-cymene based half-sandwich congener, in MDA-\n MB-231 cells under a similar protocol (protocol 1). Albeit, 2 is\n Figure 2. Comparison of in vitro IC50 for 1, 2, cisplatin and oxaliplatin in three\n more potent than the curcumin-based 1 irrespective of cell lines different cancer cell lines showcasing the highest chemotherapeutic\n or protocols (Table 2 and Figures S12-S13).[19] The results antiproliferative activity of the morphocumin based complex 2.\n\n\n Chem. Eur. J. 2025, 31, e202403695 (4 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f Table 2. Comprehensive IC50 values (in \u03bcM) for 1 and 2 in different cancer cell lines in different protocols.\n IC50 \ufffd SD[a] (\u03bcM)\n Protocol 1 Protocol 2 Protocol 3 Protocol 4\n 24 h cell seeding ! 24 h cell seeding ! 24 h cell seeding ! 24 h cell\n 4 h drug incubation ! 4 h drug incubation 24 h drug incubation seeding !\n drug removal ! ! drug removal ! ! drug removal ! 72 h drug\n 1 h photo/dark 1 h photo/dark 1 h photo/dark incubation in\n treatment in PBS ! treatment in PBS ! treatment in PBS ! dark\n 19 h cell growth in 44 h cell growth in 48 h cell growth in\n dark dark dark\n\n Cell lines Light Dark Light Dark Light Dark Dark\n\n\n\n\n Chem. Eur. J. 2025, 31, e202403695 (5 of 14)\n Chemistry\u2014A European Journal\n\n\n\n\n MDA-MB-231 1 2 1 2 1 2 1 2 1 2 1 2 1 2\n > 50 19.5 \ufffd > 50 62.3 \ufffd 4.7 \ufffd 4.4 \ufffd > 25 7.85 \ufffd 1.35 \ufffd 0.28 \ufffd 1.91 \ufffd 0.62 \ufffd 2.3 \ufffd 0.43 \ufffd\n 2.4** 4.9** 0.8** 0.3** 0.2*** 0.2** 0.02** 0.16** 0.02*** 0.5* 0.01***\n MIA PaCa-2 1 2 1 2 1 2 1 2 1 2 1 2 1 2\n > 50 5.2 \ufffd > 50 > 50 0.97 \ufffd 0.67 \ufffd 1.63 \ufffd 0.93 \ufffd 0.66 \ufffd 0.24 \ufffd 1.59 \ufffd 0.38 \ufffd 1.43 \ufffd 0.30 \ufffd\n 1.2* 0.07** 0.06** 0.5* 0.18* 0.06** 0.01 0.09*** 0.01*** 0.15** 0.02**\n PANC-1 1 2 1 2 1 2 1 2 1 2 1 2 1 2\n > 50 16.8 \ufffd > 50 > 50 2.93 \ufffd 2.8 \ufffd 4.9 \ufffd 4.9 \ufffd 1.3 \ufffd 0.73 \ufffd 3.7 \ufffd 1.1 \ufffd 2.3 \ufffd 1.1 \ufffd\n Research Article\n\n\n\n\n 0.3*** 0.4** 0.1*** 0.8** 0.02*** 0.14** 0.04** 0.15** 0.01*** 0.14** 0.1**\n\n [a] IC50 \ufffd SD are determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in normoxia (~ 15 % O2). The values show the ranges of two tailed P-values (One sample t-test, obtained\n from GraphPad Prism) corresponding to different levels of statistical significance. ns: P > 0.05, *: 0.01 \ufffd P < 0.05, **: 0.001 < P < 0.01 and ***: P < 0.001. The error bar shows standard deviation from three\n independent experiments. The error bar shows standard deviation from three independent experiments.\n doi.org/10.1002/chem.202403695\n\n\n\n\n \u00a9 2024 Wiley-VCH GmbH\n15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n Table 3. Comparative chemotherapeutic IC50 (\u03bcM) values of 1 and 2 along with Cisplatin and Oxaliplatin.\n IC50 \ufffd SD[a] (\u03bcM)\n Complex MDA-MB-231 TSI-1[b] TSI-2[c] MIA PaCa-2 TSI-1[b] TSI-2[c] PANC-1 TSI-1[b] TSI-2[c]\n\n 1 2.3 \ufffd 0.5* 6.1 8.3 1.43 \ufffd 0.15** 6.1 3.98 2.3 \ufffd 0.14** 1 1.3\n 2 0.43 \ufffd 0.01*** 32.8 44.7 0.30 \ufffd 0.02** 29 19 1.1 \ufffd 0.1** 2.2 2.8\n Cisplatin 14.1 \ufffd 0.5[50] 8.7 \ufffd 0.7** 2.38 \ufffd 0.1***\n Oxaliplatin 19.2 \ufffd 1.2[51] 3.1 \ufffd 0.2[52]\n\n [a] IC50 \ufffd SD are determined by MTT assay in normoxia (~ 15 % O2). The values show the ranges of two tailed P-values (One sample t-test, obtained from\n GraphPad Prism) corresponding to different levels of statistical significance. ns: P > 0.05, *: 0.01 \ufffd P < 0.05, **: 0.001 < P < 0.01 and ***: P < 0.001. The error\n bar shows standard deviation from three independent experiments. The error bar shows standard deviation from three independent experiments. [b] TSI-1:\n therapeutic selectivity index-1: IC50 of Cisplatin/IC50 of the complex. [c] TSI-2: therapeutic selectivity index-2: IC50 of Oxaliplatin/IC50 of the complex\n\n\n\n ma MIA PaCa-2, for comparison between a non- cancerous and\n cancerous cell line. We found that whether the drug is Natural Transition Orbitals (NTOs) for 2 predicted at 511.8 and\n incubated in the medium for 24 or 72 h the IC50 remained 534.8 nm (corresponding to the peak on the spectra), it was\n almost the same (Table S1). Furthermore, all the examined observed that the electron-hole pair transitions do not end up\n complexes were discovered to be equally toxic, if not more so, populating the Ru O \u03c3* orbitals, and hence, no ligand\n in HEK293 than in MIA PaCa-2, indicating their non-specificity dissociation upon photoirradiation (Table S5).\n (Table S1, Figures S20\u2013S21). In addition this also depicts that\n unlike the Pt drugs cisplatin and oxaliplatin the complexes are\n not susceptible to sequestration by ATP7 A which is highly Stability Comparison upon Various Stimuli Responses\n populated in HEK293.[53]\n pH stability: The stability of complexes in endosomal pH 5\n through reverse phase HPLC revealed that both the complexes\n Electrostatic Potential Maps were intact in phosphate buffer of pH 5 containing 4 mM NaCl\n up to 24 h (Figure 3a and Figures S25\u2013S26). In our previous\n The cause of exhibition of poor phototherapeutic activity of work, the [RuII(p-cymene)(morphocumin)Cl] also showed excel-\n O,O-coordinated ruthenium polypyridyl complexes is suggested lent stability in acidic pH.[19] Thus, replacing the arene motif\n to be due to the LUMO (lowest unoccupied molecular orbital) with bipyridyl did not alter the stability of these complexes.\n of such Ru(II) complexes having minimal or no contribution This signifies that if the complexes are internalized by\n from the Ru O \u03c3* orbitals. So even under photoirradiation, the endocytosis, then in the endosome the complexes remain intact\n Ru O \u03c3* orbitals remains largely unoccupied in the excited for release inside cells.\n state, thus preventing the release of the ligand for PACT type\n activity.[54] We initially conducted density functional theory\n (DFT) calculations (B3LYP/6-31G(d,p)) with water as the solvent\n (defined by the IEFPCM model) to determine the electrostatic\n potential surfaces (ESP) of 1 and 2. In Figure S22, the light blue\n area of the complexes housing the curcumin or morphocumin\n scaffold, demonstrates high electron density, while the intense\n blue region corresponding to the bipyridine motif exhibits\n reduced electron density. Both complexes exhibit similar ESP\n profiles.\n Analysis of the frontier molecular orbitals and the natural\n transition orbitals corresponding to our predicted UV-Vis\n spectra, utilizing the PM6 semi-empirical model, gave the best\n fit to the experimentally determined spectra (Figure S23) and\n the electron-hole pairs. The calculation reveals that the HOMO\n (highest occupied molecular orbital) is predominantly localized\n on the curcumin/morphocumin, while the LUMO is centered on\n the bipyridyl group (Figure S24 and Tables S2\u2013S5). The HOMO-\n LUMO bandgap for both 1 and 2 is comparable, and there is\n minimal contribution from Ru O \u03c3* orbitals in the LUMO, Figure 3. RP-HPLC chromatogram of 2 in 4 : 6 v/v DMSO and phosphate\n aligning with the literature findings. Consequently, no photo- buffer (10 mM phosphate, 4 mM NaCl) with respect to time under various\n conditions. a) Stability at pH 5 up to 24 h. b) Stability upon photo-irradiation\n release of curcumin or morphocumin from 1 and 2 respectively, (\u03bb = 400\u2013800 nm, 2.7 mW cm 2; total 10 J cm 2) up to 6 h. c) In the presence\n was observed upon irradiation with visible light. Analyzing the of 10 equiv. GSH up to 48 h. d) In presence of 5 equiv. 9-EtG up to 24 h.\n\n\n Chem. Eur. J. 2025, 31, e202403695 (6 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n Photostability: Photostability of the complexes were inves-\n tigated through UV-Vis as well as HPLC. 25 \u03bcM of each 1 and 2\n in acetonitrile was irradiated with visible light (\u03bb = 400\u2013800 nm,\n 2.7 mW cm 2, total 10 J cm 2) for 1 h (similar dose used for\n in vitro PDT) but their UV-Vis spectra remained almost unaltered\n revealing their stability upon irradiation (Figures S27 and S28).\n A similar experiment conducted by HPLC by incubating the\n complexes in 4 : 6 v/v DMSO and phosphate buffer of pH 7.4\n containing 4 mM NaCl (Figure 3b and Figures S29\u2013S30) showed\n that even up to 6 h post-irradiation (hpi) there is no photo-\n dissociation, as no new peak generated in the HPLC chromato-\n grams.\n Stability against Glutathione and 9-EtG binding: Cancer\n cells generate resistance to chemotherapy by metal complexes\n of Ru/Pt/Ir by increasing the population of soft donors like\n glutathione (GSH) exploiting the HSAB principle. GSH has a\n wide-ranging spectrum of action and extensive distribution\n because of its solubility in both water and lipids. GSH binding Figure 4. H2O2 mediated ligand release from 2. a) Cumulative plot of release\n to a drug helps to efflux it out of the cell by active transport, a of morphocumin from 2 in the presence of 200, 500 and 1000 equivalent\n H2O2. Y-axis denotes the % of intact amount of 2 and Morphocumin\n pathway of chemotherapy resistance.[55] Hence kinetic inertness (calculated from area under the peak in respective HPLC chromatograms), X-\n is an important property to fight the sequestration by soft axis denotes different time points of experiments. Solid lines are a guide to\n donors like GSH and other thiol donor based sequestration eye. b) RP-HPLC chromatogram of 2 in 4 : 6 v/v DMSO and phosphate buffer\n (10 mM phosphate, 4 mM NaCl, pH 7.4) in the presence of 200 equiv. of H2O2\n /transport proteins. Reverse phase HPLC showed no GSH bound up to 72 h showing increase in release of morphocumin from 2 with respect\n peak for 1 and 2 up to 48 h when 10 equiv. of GSH was co- to time. c) Plot of time-dependent morphocumin release from 2 in the\n incubated with respective complexes using buffer of pH 7.4 presence of 200 equiv. H2O2 by taking the area under the respective HPLC\n chromatogram from (b). Y-axis denotes the % of intact amount of 2 and\n (Figure 3c and Figures S31\u2013S32). The coordinative saturation morphocumin, while X-axis denotes different time points of experiments.\n and tight binding may be the reason for the stability. This Solid lines are a guide to eye.\n stability also makes it non-reactive towards the model nucleo-\n base 9-Ethtylguanine (9-EtG). Even 5 equiv. of 9-EtG to 1 or 2 at\n pH 7.4 showed no adduct formation (Figure 3d and Figur- dependent studies, while in tumor microenvironments continu-\n es S33\u2013S34). The above data suggests that the intact complexes ous production of H2O2 takes place.[22] Strikingly, under similar\n are active agents without needing to dissociate and dissociation conditions, 1 did not release curcumin except when the H2O2\n of the bidentate morphocumin would widen the target domain concentration was enhanced to 1000 times and then the\n for 2, since morphocumin is stable inside cell, targets lysosome complex dissociate to many uncharacterized species. Since the\n and is also photoactive. stability of curcumin is much poorer in the presence of ROS\n H2O2 mediated ligand release: Several human carcinoma compared to a normal physiological condition, it can be\n cell lines produce significant amounts of hydrogen peroxide assumed that even if it is released from 1, it would degrade.\n (H2O2) in vitro without external stimulation at a significant rate This is supported by our HPLC stability data of standalone\n unlike most normal cells. The elevated amount of H2O2, the curcumin recorded under similar concentrations of H2O2 (Fig-\n most chemically stable ROS in tumor microenvironment ure S41). The stability of morphocumin was scrutinized in\n compared to normal cells is often utilized as responsive stimuli presence of 200 equiv. H2O2 up to 24 h and it is mostly stable\n for prodrug activation or therapeutic ligand release.[56,57] A (Figure S42), unlike curcumin. So, 2 is also advantageous from\n solution of the complexes and various equivalents of H2O2 (200, the perspective of releasing morphocumin, a lysosome target-\n 500, and 1000 equiv. respectively) at 37 \u00b0C was used to ing PDT agent with much higher stability than curcumin in the\n investigate the time-dependent stability of 1 and 2 by reverse harsh environment of cancer cells. Previously, different research\n phase HPLC (Figures S35\u2013S40). Ligand release was evident for 2 groups have reported stimuli responsive release of curcumin\n (Figure 4a) but 1 dissociates to several species. Incubation of 2 derivatives from metal complexes as summarized in Table S6.\n with 200 equiv. H2O2, showed a slow release of morphocumin The data shows that curcumin is released from metal complexes\n which increased from 14 % ! 22 %! 32 % after 24 h, 48 h, and of Pt(II), Co(III) and Fe(II/III) using stimuli like visible light, GSH,\n 72 h respectively (Figure 4b, c and Figure S36). When the ascorbic acid but not H2O2 which is continuously produced in\n amount of H2O2 was elevated to 500 equiv, the percentage of cancer cells in cancer cells.\n released morphocumin enhanced up to 21 % and 36 % after GSH based release of ligand for a metal complex may be\n 24 h and 48 h, respectively (Figure 4a and Figure S38). Upon disadvantageous since when a metal complex releases curcu-\n incubating with 1000 equiv. H2O2, more than 50 % morphocu- min in the presence of excess GSH that suggests that the metal\n min released within 12 h but the morphocumin also oxidized or complex is now a potential binding site for the GSH if the metal\n degraded (Figure 4a and Figure S40). It is worth mentioning is relatively soft electrophilic centre, thus disrupting the\n that there was no auxiliary addition of H2O2 during the time- mechanism of action, which is not the case with our complex\n\n\n Chem. Eur. J. 2025, 31, e202403695 (7 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n since 2 shows significant resistance to excess GSH. The\n reactivity of 2 towards H2O2 to release morphocumin which\n unlike curcumin is stable in the ROS rich environment and also\n a phototherapeutically active compound under visible light\n irradiation makes 2 unique from its predecessors. In addition,\n the required dose for chemotherapeutic activity of these O,O-\n coordinated complex is also better than the earlier reported\n complexes depicted in Table S6.\n\n\n\n Determination of ROS Generation and Type of Photo-process\n\n The indication that 2 releases the stable morphocumin in the\n presence of hydrogen peroxide led us to evaluate the potential\n of 1 and 2 in generation of ROS. We select two distinct ROS\n indicators for each of the two potential photo-process types.\n The absorbance of 9,10-anthracenediyl-bis(methylene) dima-\n lonic acid (ABDA), a known 1O2 specific indicator, did not\n decrease upon exposure of 1 or 2 to visible light (400\u2013800 nm,\n 2.7 mW cm 2), confirming non-involvement of type II photo- Figure 5. a) Capacity factors (k) of 1 and 2 with their corresponding ligands.\n b) Correlation diagram of IC50 (in MIA PaCa-2 cells), Log P and cellular uptake\n process (Figure S43). Previously, 1 was shown to proceed via (in MIA PaCa-2 cells) for 1 and 2. c) Comparative plot of cellular uptake for 2\n type-I pathway generating ROS other than 1O2 but the exact in MIA PaCa-2 and PANC-1 cell lines. d) Lysosomal colocalization of 10 \u03bcM of\n ROS involved remains unknown.[43] We find that 1 and 2 2 in MDA-MB-231 cells using lysotracker Red DND-99 (200 nM) for observing\n co-localization along with calculated Pearson\u2019s correlation coefficient (PCC)\n produce hydroxyl radicals, a specific class of type I ROS, when value.\n exposed to photo-irradiation in PBS (5 % DMF) using a standard\n spectroscopic method that depends on the OH oxidation\n process turning the non-fluorescent HPF probe fluorescent.[58]\n The data (Figure S44) suggests OH radical (\u022eH) generation by 2 0.18), indicating greater lipophilicity for 1. Comparison with free\n upon photo-irradiation. The results obtained with 1 and 2 are curcumin (k = 5.98) and morphocumin (k = 0.26) revealed that\n similar as both proceed via type-I pathway promoting the the ligands contribute significantly to the hydrophobicity or\n generation of ROS through electron transfer. At only 1 \u03bcM dose hydrophilicity of the complexes (Figure 5a). The lower capacity\n both 1 and 2 generate ROS inside MDA-MB-231 cells converting factor of the corresponding complexes suggests that the Ru-\n the non-fluorescent DCFDA to fluorescent DCF (Figure S45) bis-bipyridyl promotes hydrophilicity. Given that there is a well-\n suggesting the O,O donor curcumin and morphocumin as a established linear relationship between log(k) and log (Po/w),[59]\n ligand are equally efficient in generating ROS but the we calibrated our system using the six reference chemicals\n disadvantage of curcumin is its instability in the presence of listed in the OECD guidelines: 1-Naphthol, Benzophenone,\n ROS unlike morphocumin, which is highly stable as discussed Phenanthrene, Diphenyl Ether, and Naphthalene. Upon fitting\n earlier. with a straight line (Figure S46), the equation obtained for the\n system (1 : 1 MeCN: H2O) is found to be:\n \ufffd\n Capacity Factor, Lipophilicity and Cellular Accumulation log Po=w \u00bc 1:90416 \ufffd log\u00f0k\u00de \u00fe 1:82532; R2 \u00bc 0:946\n\n The lipophilicity and cellular accumulation provide relevant The calculated lipophilicity shows a higher lipophilic character\n information of drug-like properties. Lipophilicity of complexes 1 of 1 compared to 2 (Table 4) in accordance with the trend in k\n and 2, determined using reverse-phase high-performance liquid values. Treatment of MIA PaCa-2 cells with equimolar concen-\n chromatography (RP-HPLC) showed that the capacity factor (k) trations (10 \u03bcM) of 1 and 2 for 6 h and analysis by Inductively\n values, which reflect the retention of compounds in the Coupled Plasma Optical Emission Spectroscopy (ICP-OES)\n chromatographic system is higher for 1 (k = 4.42) than 2 (k = showed higher cellular accumulation of 2 (almost 1.4-fold\n\n\n Table 4. Chemotherapeutic IC50, lipophilicity and cellular uptake of 1 and 2 with their corresponding ligands.\n Complexes IC50 in MIA PaCa-2 cells (\u03bcM) [72 h drug incubation] LogP Uptake in MIA PaCa-2 cells (ng/mol)\n\n 1 1.43 \ufffd 0.15 3.054 242.5 \ufffd 21\n 2 0.3 \ufffd 0.02 0.407 335.1 \ufffd 31\n Curcumin 6.15 \ufffd 0.2 3.304 ND\n Morphocumin 6.8 \ufffd 0.2 0.711 ND\n\n\n\n Chem. Eur. J. 2025, 31, e202403695 (8 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n higher than 1) (Table 4). The internalization data correlates well lysosome.[19] Hence the DNA binding mode may be a minor\n with the cytotoxicity profile, but it is important to note that pathway of action for 2.\n there is no linear correlation between lipophilicity, cellular\n uptake and cytotoxicity for 1 and 2 (Figure 5b). Based on the\n cytotoxicity profile, to ascertain if the better IC50 values were Studying the Mechanism of Action in the Presence of\n due to higher cellular uptake, we performed ICP-OES for 2 in Inhibitors\n PANC-1, in which the complex was found to be the least potent\n among three tested cell lines. The data suggested that cellular Our studies so far highlighted that whether we use protocol 4\n uptake in PANC-1 cells is 1.5-fold lesser for 2 than that in MIA or 5 for chemotherapeutic activity the results will be similar.\n PaCa-2 cells under similar conditions, which may be one of the The studies also suggest that protocol 3 may be the best choice\n major reasons for 2 showing better efficacy in MIA PaCa-2 over for measuring phototherapeutic activity in similar systems. The\n PANC-1. (Figure 5c). This also suggested that if we can improve indication from the above studies suggested that cellular\n uptake and selectivity then the potency of the complexes uptake as well as cytotoxicity profile of 2 is excellent in MIA\n would be increased (vide infra). Our next step was to find if 2 PaCa-2 cell line. We thus investigated the chemotherapeutic\n also have co-localization in lysosome since that would further mechanism of action of 2 in MIA PaCa-2 cell line by using\n elucidate if the cells were killed via lysosome degradation due various inhibitors. Since the bidentate active ligand in 2, is the\n to ROS generation inside lysosomes lysosome targeting morphocumin,[19] so we used Leupeptin, a\n known lysosomal protease inhibitor, well-documented to curb\n the activity of lysosomal hydrolase and prevent lysosome-\n Subcellular Colocalization and DNA Interaction Study mediated cell death[63] by inhibiting cysteine, serine, and\n threonine proteases.[64] The cell viability of 2 co-incubated with\n Morphocumin mostly localize in lysosome (Pearson\u2019s correlation 10 \u03bcM Leupeptin gave no difference in the IC50 values (Fig-\n coefficient (PCC) = 0.85) and so does the Ru-p-cymene complex ure 6a, Figure S50), ideally suggesting that the cell death\n (PCC = 0.65) as shown by us earlier.[19] Complex 2 however, is at rendered by the complexes are not affected by the inhibition of\n least 17 fold more active in MDA-MB-231 cells but unlike the lysosomal hydrolases. In contrast, there is almost 1.5-fold\n Ru-p-cymene-Cl congener shows lower lysosomal co-localiza- reduction in IC50 of morphocumin by co-incubation with 10 \u03bcM\n tion by confocal microscopy (PCC = 0.54) (Figure 5d). Thus, the leupeptin suggesting that free morphocumin promotes lyso-\n non-organometallic bis-bipyridyl motif conjugation with Ru(II) some-mediated cell death (Figure S51). Thus, complex forma-\n incorporates alteration in the organelle targeting ability. The tion changes the pathway of action with respect to the free\n amount of 2 accumulating in lysosome may still enable\n lysosomal disruption leading to cell death by autophagy or\n apoptosis.\n Ru(II) polypyridyl complexes and curcumin derivatives have\n shown affinity towards DNA.[60] Previously, Ru bis-bipyridyl\n curcumin complex (1) and its analogs were found to be DNA\n groove-binders.[43,61] This information guided us to investigate\n nuclear localization of complex 2. In MDA-MB-231 cell line, the\n result shows a marginal localization of 2 in nucleus (PCC = 0.27)\n (Figure S47). The nuclear colocalization is minimal; however, we\n still conducted DNA interaction studies to determine if there is\n a possibility of interaction. Since Ru polypyridyl complexes and\n Ru-polypyridyl-curcumin scaffolds are reported for binding with\n DNA through multiple interaction modes.[4,44,61]\n 1 and 2 showed hypochromism of calf thymus DNA\n absorption indicating interaction between the complex\u2019s elec-\n tronic states and the stacked DNA base pairs (Figure S48a-b).\n This may be because the partially filled \u03c0* orbital of 1 and 2\n comes in proximity with the DNA\u2019s \u03c0 orbitals, lowering Figure 6. a) Cytotoxicity of 2 in MIA PaCa-2 cell line in presence of various\n transition probabilities, evidenced by hypochromism.[62] inhibitors, Leupeptin (10 \u03bcM) as lysosomal protease inhibitor; N-acetylcys-\n teine (5 mM) as ROS suppressor; 3-methyl adenine (100 \u03bcM) as autophagy\n Fluorescence intensity of 2 also increased upon sequential inhibitor; Ferrostatin-1 (60 \u03bcM) as Ferroptosis inhibitor; Necrostatin-1 (60 \u03bcM)\n addition of CT-DNA addition suggests that interaction with DNA as Necroptosis inhibitor. The figure shows the ranges of P-values (Two\n may be restricting the free rotation of 2 in solution, enhancing sample t-test) corresponding to different levels of statistical significance. ns:\n P > 0.05, *: 0.01 \ufffd P < 0.05, **: 0.001 < P < 0.01 and ***: P < 0.001. The error\n the fluorescence (Figure S48c).[62] However, it must be noted bar shows standard deviation from three independent experiments. b)\n that 2 co-localizes more in lysosome than in nucleus and in Change in mitochondrial membrane potential upon 24 h treatment of 1 and\n presence of ROS, 2 would release morphocumin in cell and the 2 (1 and 2 \u03bcM) in light and dark. c) Cell cycle phase arrest of 2 (0.5, 1, 2 \u03bcM\n for 24 h) in light and dark. d) Induction of apoptosis by 1 and 2 (1 and 2 \u03bcM\n released morphocumin is known to localize highly in for 24 h) in chemotherapeutically in MIA PaCa-2 cell lines as analyzed\n through Flow cytometry.\n\n\n Chem. Eur. J. 2025, 31, e202403695 (9 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n morphocumin. Complex 2 also does not kill by autophagy as the cell cycle. MIA PaCa-2 cells were treated with various doses\n indicated by the unchanged IC50 values in presence of 100 \u03bcM of 1 and 2 in the presence and absence of light. Both 1 and 2\n of standard autophagy inhibitor 3-methyladenine (Figure 6a exhibit arrest in Sub G0 phase upon photo-irradiation while\n and Figure S50). The IC50 of 2 also did not change in presence interfering with the G2/M phase in dark (Figure 6c and\n of 60 \u03bcM of each Ferrostatin-1 and Necrostatin-1 suggesting Figures S55\u2013S56). There are several factors which may influence\n Ferroptosis or necroptosis respectively are not involved in cell the arrest of cell cycles by a compound, one of them is certainly\n death (Figure 6a and Figure S50). the specificity and toxicity of the drug. Drugs that arrest in Sub\n The intracellular ROS generation study reveals the potential G0 phase may be more selective for cancer cells and can spare\n of 2 to induce ROS irrespective of light or dark. So, the normal cells that are in the resting state, as they can target cells\n cytotoxicity of 2 was investigated in the presence of N- that have abnormal signals or damages.[69] On the other hand,\n acetylcysteine (NAC), a well-known ROS inhibitor and suppres- drugs that arrest in G2/M phase may affect both cancer and\n sor. 2 co-incubated with 5 mM NAC gave two-fold poor IC50 normal cells, as they can interfere with the normal cell cycle.[69,70]\n (Figure 6a and Figure S50). NAC plays a significant role in It is also reported for several photoactive drugs to have\n reducing oxidative stress by scavenging ROS like, peroxide and different cell cycle phase arrest in the presence and absence of\n hydroxyl radicals to prevent cellular damage. It also promotes light.[71] However, in our case, such differences were not\n glutathione synthesis, mitochondrial protection and prevent anticipated, since we observed minimal alteration in dose\n apoptosis induced by oxidative stress.[65] Thus, co-incubating 2 response in the presence and absence of light. However, the\n with NAC leading to poor IC50 suggests a possible role of 2 in cell cycle arrest and the mitochondrial depolarization data\n inducing oxidative stress through ROS generation. suggest that in spite of the toxicity dosage being similar in the\n presence and absence of light there exists a difference in\n pathway of action.\n Investigating the Mechanism of Action: Cyclic Voltammetry, Annexin-V PE and 7AAD were used in the cell death assay,\n NAD + Interaction, JC-1, Cell Cycle Arrest and Apoptosis which demonstrated that both 1 and 2 cause apoptosis in a\n dose-dependent manner (Figure 6d and Figure S57). Accord-\n A compound may help accumulation of ROS in several ways ingly, our findings imply that the ROS produced by the\n which include disrupting the mitochondrial function or reacting complexes kill through the intrinsic mechanism of apoptosis\n with the molecular oxygen in cells. The complex may be redox mediated by the mitochondria.\n active in cellular conditions. Thus, we investigated 2 for redox\n activity in dark. The data showed that the oxidation potential of\n 2 in acetonitrile is 0.47 V vs. SCE (0.07 V vs. Ferrocene; 0.71 V vs. Encapsulation in Glucose Functionalized Polymeric\n NHE) (Figure S52). It is suggested that Ru-polypyridyl complexes Nanoparticles\n having redox potential value in the range of 0.4\u20130.6 V vs. SCE\n are found to be most cytotoxic.[66] The value of redox potential One of the hallmarks of pancreatic ductal adenocarcinoma\n of 2 with respect to NHE is close to the biological range of (PDAC) is dramatically enhanced glycolytic flux, characterized\n \ufffd 0.5 V.[67] The ability of 1 and 2 to catalyze the reduction of by \u2018Warburg effect\u2019, and this is also true for many other\n NAD + to NADH was scrutinized in the presence of formate as a cancers.[72] In similarity with many other cancers, in PDACs to\n hydride donor.[68] Both 1 and 2 (2 \u03bcM) co-incubated with NAD + fulfill the requirement of glucose and its transport across\n (100 \u03bcM) in the presence of formate (300 \u03bcM) showed no plasma membrane GLUT1, also known as solute carrier family 2\n reduction of absorbance at \u03bb340 nm excluding the possibility of facilitated glucose transporter member 1 (SLC2 A1), is\n NAD + reduction to NADH (Figure S53) by 2. overexpressed.[73,74] Studies analyzing GLUT-1 expression in 53\n Next, we looked into the possible change of mitochondrial pancreatic cancer tissues found that overexpression of GLUT-1\n membrane potential since 2 show accumulation of ROS which was associated with poor prognosis and adverse clinicopatho-\n may be due to disruption of the mitochondrial function. During logical features, including increased tumor size, higher max-\n the course of several different cell death modes, an event that imum standardized uptake value (SUVmax), lymph node meta-\n begins promptly is the depolarization of the mitochondrial stasis, advanced clinical stage, and elevated Ki-67\n membrane potential (MMP). MIA PaCa-2 cells were treated with expression.[74,75] In spite of the excellent cytotoxicity, 2 displays\n 1 \u03bcM and 2 \u03bcM dose of both 1 and 2 in the presence as well as low chemotherapeutic selectivity index (IC50 in normal cell/IC50\n absence of photo-stimuli. Significant dose dependent and in cancer cell) thus to enhance selectivity and make delivery\n stimuli dependent depolarization of MMP by 1 and 2, showed more feasible we employ a glucose conjugated polymer with\n elevation of JC-1 monomer\u2019s green emission intensity compared the capacity to form micelles that encapsulate and deliver 2\n to the red emitting aggregates present in healthy mitochondria. more selectively to cancer cells.[40,41]\n Both 1 and 2 induced greater depolarization of MMP upon We noted that establishing a suitable interaction between\n photo-irradiation compared to dark condition and 2 exhibited the glucose units of the polymer and GLUTs is feasible when\n pronounced effectivity in all cases compared to 1 (Figure 6b the polymeric nanocarriers are structured in a manner that\n and Figure S54). ensures the retention of glucose segments within their outer\n Then, the cell cycle analysis in MIA PaCa-2 cells were shell. Moreover, to attain optimal drug encapsulation within the\n performed to know if there is arrest in any particular phase of nanocarriers, it is imperative to have balanced hydrophilic and\n\n\n Chem. Eur. J. 2025, 31, e202403695 (10 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n hydrophobic segment in the polymer. Therefore, a glucose- (DLS) studies. The CAC of DP, determined using Nile red (NR) as\n based polymer was synthesized utilizing the reversible addition- a hydrophobic fluorescent probe[77] is approximately 30.1 \u03bcg/mL\n fragmentation chain-transfer (RAFT) polymerization technique, in aqueous medium (Figure 7b). Additionally, the morphology\n ensuring controlled chain growth and precise incorporation of of these micelles was further validated by field emission\n hydrophobic and hydrophilic segments within the polymer. scanning electron microscopy (FESEM) and transmission elec-\n Initially, the glucose-based monomer Ac G-EMA (Figure 7a tron microscopy (TEM) analyses, with the estimated sizes of DP\n and Figure S58) was synthesized according to previous from TEM and FESEM images being 76.1 \ufffd 9.8 nm and 71.5 \ufffd\n literature[76] using \u03b2-D-glucose pentaacetate and 2-hydroxyethyl 15.3 nm, respectively.\n methacrylate (HEMA). The Ac G-EMA was then copolymerized Once the glucose-based copolymer was synthesized, we\n with hydrophobic methyl methacrylate (MMA) via the RAFT proceeded to encapsulate complex 2 in DP to create drug-\n polymerization method, employing 4-cyno-4-(dodecylsulfa- loaded glucose-based nanoparticles, labeled DP@2. We calcu-\n nylthiocarbonyl) (CDP) as the chain-transfer agent (CTA) and lated the drug loading efficiency (DLE) and drug loading\n 2,2-azobisisobutyronitrile (AIBN) as the initiator. During the content (DLC) of the nanoparticles,[78] finding them to be 46.7 %\n polymerization, the molar ratio of [Ac G-EMA]/[MMA]/[CDP]/ and 7.0 %, respectively. Notably, the inclusion of 2 in DP did not\n [AIBN] was maintained as 20/10/1/0.1 (Figure S59). The resulting alter the hydrodynamic diameter (Dh) as measured in PBS, with\n copolymer, P(Ac G-EMA-co-MMA), was characterized by using DP and DP@2 showing Dh values of 82.9 \ufffd 7.2 nm (PDI 0.599)\n 1\n H NMR and size exclusion chromatography (SEC) analysis. The and 75.2 \ufffd 6.2 nm (PDI 0.635), respectively (Figure 7c). To\n number average molecular weight (Mn,NMR) of the polymer was further investigate the morphology of DP@2, TEM and FESEM\n calculated from NMR analysis with respect to the end-group analyses were conducted, revealing sizes of 88.2 \ufffd 5.2 nm and\n protons (COOHCH2CH2 ) of CDP at 2.4\u20132.6 ppm, yielding a 64.2 \ufffd 13.2 nm, respectively (Figure 7d\u2013g). The SEM-EDS spec-\n value of 8000 g/mol. This value is in close agreement with the trum of DP@2 confirms the metal complex encapsulation\n theoretical molecular weight (Mn,theo), which was calculated (Figure S61). It is well-known that micelles smaller than 100\u2013\n using the following equation: Mn,theo = (([monomer]/[CDP] \u00d7 150 nm are less likely to be cleared by the reticuloendothelial\n average molar mass of monomer \u00d7 conv.) + (molar mass of system, making them suitable for drug delivery applications.[79,80]\n CDP)). SEC analysis provided the number average molecular Therefore, we examined the efficacy of DP@2 in cancer therapy,\n weight (Mn,SEC) and dispersity (\u0110) of the polymer, with given its ability to release 2 within the cellular environment\n poly(methyl methacrylate) (PMMA) as a standard (Figure S60). through esterase-mediated micellar degradation.\n The polymer\u2019s characterization is summarized in Table S7. To exploit the altered metabolism of cancer cells charac-\n Finally, the acetyl groups of P(Ac G-EMA-co-MMA) were depro- terized by accelerated glycolysis (Figure 8a), we assessed the\n tected to yield the desired glucose-conjugated copolymer (DP) cytotoxicity of DP and DP@2 in MIA PaCa-2 and HEK 293 cell\n (Figure 7a and Figure S59). In an aqueous medium, the lines. Results showed that DP is non-toxic at doses up to\n micellization of DP was confirmed through critical aggregation 0.5 mg/mL in both cell lines (Figure S62). We also evaluated the\n concentration (CAC) measurement and dynamic light scattering IC50 values after a 24 h treatment to gauge the true effective-\n\n\n\n\n Figure 7. a) Schematic representation of synthesis of Ac G-EMA, P(Ac G-EMA-co-MMA) and DP. b) CAC plot of DP in water; c) DLS plot of DP and DP@2 in\n PBS; TEM images of d) DP and e) DP@2. Scale bar = 500 nm; FESEM images of f) DP and g) DP@2. Scale bar = 100 nm.\n\n\n Chem. Eur. J. 2025, 31, e202403695 (11 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 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\nChemistry\u2014A European Journal doi.org/10.1002/chem.202403695\n\n\n (Figures 8d and Figure S63). In order for the above conclusion\n to be concrete we needed to ensure that low glucose\n concentration does not significantly alter cell survival. Thus, we\n carried out a control study varying glucose concentration with\n no drug, with DP (0.5 mg/mL) and DP@2 (5 \u03bcg/mL and 10 \u03bcg/\n mL). The 72 h treatment study confirmed that cell survival is\n similar in both high and low glucose media (Figure 8c) and due\n to higher uptake taking higher amount of DP@2 enhances\n killing ratio.\n\n\n Conclusions\n\n The study highlights the effectiveness of a Ru(II) bis-bipyridyl\n morphocumin complex (2) encapsulated within a glycopolymer,\n termed as DP@2, which exhibits high selectivity towards various\n cancer cell lines compared to non-cancerous HEK 293 cells.\n Complex 2 shows significant chemotherapeutic potential,\n particularly against the PDAC, MIA PaCa-2 as evidenced by its\n low IC50 value of approximately 300 nM. Noteworthy drug-like\n properties of 2 include its stability under physiological and\n endosomal pH conditions, as well as its stability under visible\n light and in the presence of glutathione. The high selectivity of\n the DP@2 delivery assembly suggests that these non-selective\n Figure 8. a) Schematic representation of Warburg effect and its consequen- yet highly efficient Ru(II) polypyridyl complexes can be molded\n ces in pancreatic cancer. b) Enhancement of cancer selectivity upon\n encapsulation: IC50 of 2 and DP@2 in MIA PaCa-2 and HEK 293 cell lines into selective and effective promising chemotherapeutic agents.\n cultured in high glucose (25 mM) medium following protocol 5 (24 h drug Our detailed protocol analysis for phototherapy vs. chemo-\n incubation followed by drug removal and 48 h further incubation). c) therapy assays advises caution in using the proper protocol for\n Comparative cytotoxicity plot of DP@2 in MIA PaCa-2 cells culturing in high\n (25 mM) and low (5.5 mM) glucose DMEM culture medium. Cytotoxicity is phototherapy (e. g., Protocol 3) to accurately distinguish their\n done by following protocol 5. IC50 \ufffd SD are determined by MTT assay in activity from observed dark toxicity across various cell lines. The\n normoxia (~ 15 % O2). The statistical significance (P) of the data is < 0.001. morphocumin release in presence of excess H2O2 and\n The error bar shows standard deviation from three independent experi-\n ments. d) Cumulative bar diagram of % of MIA PaCa-2 cell viability for DP enhancement of reactive oxygen species (ROS) by 2 inside\n and DP@2 as well as untreated control in high (25 mM) and low (5.5 mM) cellular environment suggest a possible synergistic domino\n glucose medium. Each experiment was done in triplicate. The figure shows effect releasing more morphocumin bringing in multiple path-\n the ranges of p-values corresponding to different levels of statistical\n significance. ns: P > 0.05, *: 0.01 \ufffd P < 0.05, **: 0.001 < P < 0.01 and ***: ways of action via lysosome and mitochondrial function\n P < 0.001. The error bar shows standard deviation from three independent disruption to induce apoptotic cell death. The suitability of the\n experiments. glycopolymer as a delivery vehicle due to excellent loading of 2\n suggests a good compatibility between the DP and 2.\n Importantly, the proof-of-concept for selectivity is shown\n ness of chemotherapy. DP@2 exhibited an IC50 of 6.6 \ufffd 0.02 \u03bcg/ through glucose functionalization, enhancing selectivity by\n mL in MIA PaCa-2 cells, while in HEK 293 cells, the IC50 was approximately 9-fold towards PDAC compared to a fast-growing\n 58.35 \ufffd 1.4 \u03bcg/mL, demonstrating nearly a 9-fold selectivity for non-cancerous cell line even in high glucose concentration\n cancer cells (Figure 8b and Figure S63). which is further increased by 5-fold more under glucose\n To further understand the role of glucose-functionalized deprivation. This underscores the potential of glycopolymer\n polymeric encapsulation, we examined the receptor-mediated delivery vehicle-based DP@2 for targeting cancer cells with\n engulfment efficiency of DP@2. We prepared DMEM medium elevated glycolysis rates. Overall, this work broadens the\n with low glucose content. Typically, the standard DMEM culture application of Ru complexes with stable curcumin derivatives\n medium contains a high glucose concentration of 25 mM. In and demonstrates the feasibility of using RAFT-based glycopol-\n this study, we determined the chemotherapeutic IC50 values of ymers to deliver complexes like 2, which can release the O,O\n DP and DP@2 by culturing them in a low glucose (5.5 mM) donor ligand morphocumin within lysosomes in response to\n medium. Since cancer cells have a high demand for glucose, specific stimuli, such as hydrogen peroxide, produced in cancer\n culturing them in glucose-deprived medium should prompt cells.\n glucose-functionalized DP or encapsulated DP@2 to enter the\n cancer cells via glucose receptors through endocytosis to meet\n their glucose requirements. As expected, the IC50 of DP@2\n decreased to 1.2 \ufffd 0.04 \u03bcg/mL, which is 5.5 times more effective\n than its IC50 in high glucose medium under similar conditions\n\n\n Chem. Eur. J. 2025, 31, e202403695 (12 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f 15213765, 2025, 8, Downloaded from https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/chem.202403695 by Lomonosov Moscow State University, Wiley Online Library on [12/05/2026]. 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Accepted manuscript online: November 30, 2024\n Martin, J. Chang, A. F. Hezel, S. R. Perry, J. Hu, B. Gan, Y. Xiao, J. M. Asara, Version of record online: December 10, 2024\n\n\n\n\n Chem. Eur. J. 2025, 31, e202403695 (14 of 14) \u00a9 2024 Wiley-VCH GmbH\n\f", "pages_extracted": 14, "text_length": 104848}