Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma.

{"full_text": " pubs.acs.org/JACS Article\n\n\n\n Microtubule-Targeting NAP Peptide-Ru(II)-polypyridyl Conjugate As\n a Bimodal Therapeutic Agent for Triple Negative Breast Carcinoma\n Atin Chatterjee, Sandip Sarkar, Sangheeta Bhattacharjee,\u2207 Arpan Bhattacharyya,\u2207 Surajit Barman,\n Uttam Pal, Raviranjan Pandey, Anitha Ethirajan,* Batakrishna Jana,* Benu Brata Das,*\n and Amitava Das*\n Cite This: J. Am. Chem. Soc. 2025, 147, 532\u2212547 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n\n\n ABSTRACT: Triple-negative breast cancer (TNBC) poses signifi-\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:24:53 (UTC).\n\n\n\n\n cant treatment challenges due to its high metastasis, heterogeneity,\n and poor biomarker expression. The N-terminus of an octapeptide\n NAPVSIPQ (NAP) was covalently coupled to a carboxylic acid\n derivative of Ru(2,2\u2032-bipy)32+ (Rubpy) to synthesize an N-stapled\n short peptide-Rubpy conjugate (Ru-NAP). This photosensitizer (PS)\n was utilized to treat TNBC through microtubule (MT) targeted\n chemotherapy and photodynamic therapy (PDT). Ru-NAP formed\n more elaborate molecular aggregates with fibrillar morphology as\n compared to NAP. A much higher binding affinity of Ru-NAP over\n NAP toward \u03b2-tubulin (KRu\u2011NAP: (6.8 \u00b1 0.55) \u00d7 106 M\u22121; KNAP: (8.2\n \u00b1 1.1) \u00d7 104 M\u22121) was observed due to stronger electrostatic\n interactions between the MT with an average linear charge density of\n \u223c85 e/nm and the cationic Rubpy part of Ru-NAP. This was also\n supported by docking, simulation, and appropriate imaging studies. Ru-NAP promoted serum stability, specific binding of NAP to\n the E-site of the \u03b2III-tubulin followed by the disruption of the MT network, and effective singlet oxygen generation in TNBC cells\n (MDA-MB-231), causing cell cycle arrest in the G2/M phase and triggering apoptosis. Remarkably, MDA-MB-231 cells were more\n sensitive to Ru-NAP compared to noncancerous human embryonic kidney (HEK293 cells) when exposed to light\n (LightIC50Ru\u2011NAP[HEK293]: 17.2 \u00b1 2.5 \u03bcM, compared to LightIC50Ru\u2011NAP[MDA-MB-231]: 32.5 \u00b1 7.8 nM, DarkIC50Ru\u2011NAP[HEK293]:\n > 80 \u03bcM, compared to DarkIC50Ru\u2011NAP[MDA-MB-231]: 2.9 \u00b1 0.5 \u03bcM). Ru-NAP also effectively inhibited tumor growth in MDA-MB-\n 231 xenograft models in nude mice. Our findings provide strong evidence that Ru-NAP has a potential therapeutic role in TNBC\n treatment.\n\n\n \u25a0 INTRODUCTION\n Triple-negative breast cancer (TNBC) is a highly metastatic,\n dividing cells in mammalian cells. These constitute the highly\n dynamic mitotic spindle and this accounts for the extreme\n heterogeneous breast cancer with impaired expression of sensitivity toward therapeutic inhibitors.6 MT targeting agents\n (MTAs) are a type of vascular disrupting agents (VDAs).5\n estrogen (ER), progesterone (PR), and human epidermal\n Chemotherapeutic drugs that bind to the colchicine site of\n growth factor receptor 2 (HER2).1,2 Poor prognosis and its\n tubulin target tumor vasculature to induce blood vessel\n low response to therapeutics add to therapeutic challenges\n disruption are used in clinical trials.13,14 Certain vinca alkaloids\n compared to non-TNBC cases.3 Recent reports confirm that\n and taxanes are part of the FDA-approved drugs for treating\n microtubule (MT) cytoskeletons are validated targets for\n hematological malignancies and solid tumors.15,16 Taxol\n breast cancer therapy.4,5 MTs are associated with critical\n belongs to the taxane family and is being used for treating\n cellular functions including mitosis, cell signaling, intracellular\n early-stage, advanced, and metastatic breast cancer.17 A 3.5-\u00c5\n trafficking, and angiogenesis.6 Any molecule that can modulate\n resolution electron crystallography structure of the tubulin\n the MT dynamics would induce the spindle checkpoint,\n arresting cell-cycle progression at mitosis and subsequent cell\n death. MT stabilizers stimulate the assembly of purified tubulin Received: August 27, 2024\n and shift the equilibrium of tubulin polymer from the soluble Revised: December 12, 2024\n to the polymerized form.7\u22129 This leads to consequential Accepted: December 12, 2024\n disruption of MT dynamics and mitotic arrest of cancer cells, Published: December 26, 2024\n which has been effectively utilized as a treatment strategy for\n cancer.6,10\u221212 MTs are present both in interphase cells and in\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/jacs.4c11820\n 532 J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\ndimer bound to taxol reveals that taxol binds stoichiometrically reactions. Here, the triplet state (T1) of the PS interacts with\nand specifically to the \u03b2-tubulin subunit in MTs.15 A few of the 3\n O2 to form 1O2, a potent ROS.40 Therefore, combining PDT\nmost recent and effective cancer chemotherapeutic drugs are and chemotherapy is crucial to counterbalance each other\u2019s\nderived from natural compounds that target MTs and disrupt limitations.41 Considering this, we have utilized NAP peptide\nthe normal function of the mitotic spindle.18 Site-specific conjugated Ru(2,2\u2032-bipy)32+ (Rubpy) derivative (Ru-NAP) as\nbinding of such molecules, either to soluble tubulin or to the a potent bimodal chemotherapeutic and PDT agent for killing\nMT governs the efficacy of these drugs as chemotherapeutic TNBC cells (Scheme 1). Ru-NAP conjugate is more efficient\nagents.11 The major issues that limit the clinical efficacy of\ntaxanes as the MTAs are low solubility and thus bioavailability, Scheme 1. (a) Cartoon Representation of the Synthetic\na relatively short circulation time t1/2 (\u223c1 h), inability to cross Methodology Adopted for the Synthesis of Ru-NAP, (b)\nthe blood-brain barrier, high systemic toxicity (e.g., neutrope- Molecular Structure of Ru-NAP,and (c) Schematic\nnia, neurotoxicity, genotoxicity, cardiac toxicity, repression of Representation of Microtubule Targeted Chemotherapeutic\nbone marrow, etc.) and the onset of multidrug resistance and PDT Action Induced by Ru-NAP\nmechanisms.17,19 These challenges offer a distinct scope for\nadopting an alternate strategy in developing MTAs for\nimproved clinical efficacy toward cancer therapy.\n NAPVSIPQ (Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln; all amino\nacids are in L- form) abbreviated as NAP is a neuroprotective\noctapeptide derived from activity-dependent neuroprotective\nprotein (ADNP). Affinity chromatography identified the \u03b2III-\ntubulin as a major NAP-binding protein.20 Presumably,\nbinding of NAP to \u03b2III-tubulin induces a conformational shift\nto facilitate tubulin polymerization with the E-site of the \u03b2III-\ntubulin, which is preferentially occupied by guanosine\ntriphosphate (GTP) rather than guanosine diphosphate\n(GDP).21 As discussed above, MTAs could be a part of an\neffective strategy for treating breast cancer. TNBC remains a\nchallenging subtype of breast cancer, and there is distinct scope\nto address the unmet needs to improve the outcome of TNBC\ntherapeutics. With this aim, we have used the \u03b2III-tubulin-\nspecific binding of the NAP peptide in demonstrating a proof-\nof-concept of a microtubule-specific photosensitizer (PS) for\nthe bimodal killing of the TNBC cells using chromophore-\nassisted light inactivation (CALI) concept.22,23 Specific\nbinding of the NAP peptide conjugated to a substitution\ninert Ru(II)-polypyridyl moiety (a singlet oxygen (1O2)\ninitiator) to the cytoskeleton protein allows this conjugate to\nqualify as CALI-based agent while addressing the limitations of\nsystemic toxicity induced by conventional PDT-agents through\nindiscriminate intracellular reactive oxygen species (ROS)\ngeneration.24\u221227 Short lifetime (\u223c6 \u03bcs) and diffusion length\n(<50 nm) for 1O2 in human physiology enable a highly\nlocalized spatiotemporal activity to minimize the possibility of\nindiscriminate systemic toxicity.23,28\u221232 Importantly, literature\nreports suggest that CALI-based PDT agents are rarely in stabilizing MT by favoring tubulin polymerization. Live cell\nexploited for in vivo studies.31,32 Antibody-drug conjugates microscopy confirms the targeting ability of Ru-NAP to the\n(ADCs) have also been conceived to achieve organelle/tissue- MT in TNBC (MDA-MB-231) cells. Thus, it causes certain\nspecific release of cytotoxic drugs to avoid nonspecific toxicity in the dark through the binding of Ru-NAP fibers to\nbiodistribution and undesired systemic toxicities. However, the tubulin/MT, followed by disruption of the MT network\nthe major shortcomings of the first- and second-generation which signifies its\u2019 chemotherapeutic effect. Moreover, in vitro\nADCs include the immunogenic responses and fast clearance and intracellular studies confirm that Ru-NAP also exhibits\nin circulation, apart from other drug-design issues.33\u221237 superior phototoxicity compared to the individual compo-\nArguably, small molecular weight peptide-drug conjugates nents, Rubpy or NAP. The higher toxicity toward live MDA-\nhave the advantages of higher drug loading and enhanced MB-231 cells accounts for the in situ generation of 1O2 for\ntissue penetration capacity, degradation without triggering any inducing PDT, along with the disruption of the MT network\nimmunogenic responses in physiology, stability toward and G2/M cell cycle arrest to induce chemotherapeutic effect.\nproteolysis, and facile synthesis.38,39 Recently, Kornienko, Importantly, the Hill plot supports a cooperative effect for Ru-\nBonnet, and their co-workers have demonstrated the efficacy NAP as a PDT agent. Control studies confirm that Ru-NAP is\nof photoactivated chemotherapy in A549 xenografts in nude benign to normal healthy human embryo kidney (HEK293)\nmice through the in situ release of an MT-targeting drug, cells under dark and light irradiation compared to MDA-MB-\nrigidin.19 231 cells. The higher efficacy of Ru-NAP toward live MDA-\n The PDT efficiency is compromised by the hypoxic feature MB-231 cells is also reflected in the significantly higher\nof tumors, especially for the PDT agents that induce Type II inhibition of tumor growth in the MDA-MB-231 tumor\n 533 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\nxenograft model with insignificant in vivo toxicity. This report mixture (1:1) with the addition of an equivalent amount of\ndemonstrates the proof of concept of MT-targeted and serum- Na2CO3. The solution was allowed to reflux at 100 \u00b0C for 8 h\nstable (due to the small size and the N-stapled termini of the followed by the addition of a saturated aq. solution of KPF6 to\nNAP peptide) PS as an effective bimodal (chemotherapeutic precipitate the crude complex of Ru-COOH. The precipitate\nand PDT) agent for inhibiting tumor growth. Such examples was purified by column chromatography and characterized\nare rather scarce in contemporary literature.9,42\u221247 using 1H NMR and ESI-MS (Figures S4\u2212S7). Then, Ru-\n\n\u25a0 RESULTS AND DISCUSSION\n Design Strategy of Ru-NAP. Literature reports suggest\n COOH was converted to an acid-chloride intermediate (Ru-\n COCl) and conjugated to a NAP peptide-bound rink amide\n resin by the SPPS method.60 This resin-attached Ru-NAP was\nthat the lifetime (T1/2) of 1O2 in the cytoplasm is about 1 \u00d7 further subjected to TFA cleavage and cold ether treatment to\n10\u22127 s with a maximum radius of reactive action of 30 nm.39 yield crude Ru-NAP. The crude Ru-NAP was purified using\nWhile, at low bacterial concentrations during photodynamic reverse phase HPLC (C18 column and acetonitrile\u2212water\ninactivation of bacteria, T1/2 for 1O2 is reported to be 6 \u00b1 2 \u03bcs gradient) and characterized using HPLC, 1H NMR, and ESI-\nwith a diffusion length of <50 nm.48 However, the efficient Ms (Schemes 1, S2 and Figures S8\u2212S10). Pyrene is known to\ntherapeutic performance of the Type II pathway is hindered by increase the hydrophobicity and self-assembled efficacy of the\nan insufficient supply of molecular oxygen (O2) in the tumor NAP. We also synthesized a pyrene-conjugated NAP peptide\nmicroenvironment (TME) because of hypoxic tumor cells. derivative (Py-NAP, Scheme S3) for our control studies using\nMicrotubules (MTs) are also involved in the manipulation of the SPPS method. Detailed synthetic procedures and\ntumor hypoxia. Hypoxia-inducible factor 1 (HIF-1), being characterization data required to ensure the desired purity of\noverexpressed in the TME regulates several target genes Py-NAP are provided in the Supporting Information. After\ninvolved in tumor angiogenesis, matrix metabolism, apoptosis, synthesis, Py-NAP was purified using reverse phase HPLC\nand glycolysis leading to tumor hypoxia.49 Tumor angiogenesis (C18 column and acetonitrile\u2212water gradient) and charac-\nis triggered by hypoxia via activation of the HIF-1 pathway, terized using HPLC, ESI-Ms (Figures S11 and S12). We\nand HIF-1 utilizes microtubules as tracks for their nuclear further synthesized 4-nitro-2,1, 3-benzoxadiazole (NBD)\ntranslocation and transcription followed by hypoxic induction. conjugated NAP (NBD-NAP; Schemes S4 and S5) for\nThus, targeting and disruption of MT dynamics serve as potent checking the aggregation inside the cell and characterized\nbarriers for tumor angiogenesis through the downregulation of using HPLC, 1H NMR, and ESI-Ms (Figures S13\u2212S16).\nthe HIF-1 pathway, which is supposed to increase the efficacy Photophysical Studies of Ru-NAP. After successful\nof the Type II PDT agents. This motivated us to utilize the synthesis, we performed the photophysical studies of the Ru-\nRubpy derivative as an MT-targeted Type II PDT agent in the NAP and compared it with the commercially available PDT\nhypoxic tumor microenvironment.43,50\u221253 Rubpy derivatives agent Rubpy. The electronic and fluorescence spectra of\nare an attractive choice as a PS primarily for the high quantum Rubpy and Ru-NAP were recorded. A distinct red shift in the\nyield for a relatively long-lived transient triplet state (\u223c100 ns) respective absorption (461 nm, \u0394\u03bbAbs = 7 nm) and emission\nthat is crucial for 1O2 generation, photochemical stability, (651 nm, \u0394\u03bbEms = 37 nm) maximum was observed for Ru-\namphiphilicity, stability toward photobleaching, insignificant NAP as compared to Rubpy in PBS buffer (pH 7.4; Figure\ndark toxicity/mutagenicity and facile cellular internaliza- S17). The relative emission quantum yield (\u03a6EmRu\u2011NAP) for Ru-\ntion.54,55 Importantly, the COO\u2212-terminal tails of several NAP was evaluated as 0.70 (Rubpy as a reference) in\nacidic amino acid residues of \u03b1- and \u03b2-tubulins are located on deionized water, saturated with O2 (Figure S18). Considering\nthe outer surface of MTs, and this accounts for an overall the substitution of one 2,2\u2032-bipy with NAP functionalized 2,2\u2032-\nnegatively charged surface. These carboxy-terminal tails, bipy through an amide linkage in Ru-NAP, these redshifts are\nmostly of glutamate side chains on \u03b1- and \u03b2-tubulins of anticipated. Luminescence lifetimes of the Ru-NAP (291.7 \u00b1\nMTs, are the key sites of interactions for many MT-binding 1.002 ns) and Rubpy (366 \u00b1 1.42 ns) using \u03bbEx = 450 nm;\nproteins or peptides.56 The estimated electric field of a single \u03bbEmRu\u2011NAP = 651 nm and \u03bbEmRubpy = 614 nm in aq. PBS buffer\nMT is 52.2 electronic charges (e) per dimer.57,58 These would (pH = 7.4) at room temperature (RT) was evaluated (Figure\nfurther favor interaction between the cationic Rubpy part of S19 and Table S1). Typically, such molecules with an extended\nRu-NAP, apart from the binding of NAP to the MTs. luminescence lifetime are better suited for use as a PDT\n Synthesis and Characterization of Ru-NAP and the agent.26,64\u221267\nControl Molecules. With these rationales, NAP and Ru-NAP Docking and Simulations Studies. We performed\nwere synthesized following procedures that are elaborated in docking studies to check the validity of our design rationale\nthe Supporting Information. NAP was synthesized following and also to understand the possible binding site for Ru-NAP to\nthe standard Hydoxybenzotriazole (HBTU) coupling protocol the tubulin dimer. Both NAP and Ru-NAP showed\nof the solid phase peptide synthesis (SPPS) method (Scheme thermodynamically favorable binding interactions with tubulin,\nS1).59 The crude peptide was purified using reverse-phase as reflected in the negative binding free energies (Tables S2\u2212\nhigh-performance liquid chromatography (HPLC) with C18 S4). The average binding energy values obtained for NAP and\ncolumn and acetonitrile\u2212water gradient, and purified NAP was Ru-NAP are \u22126.77 \u00b1 0.61 and \u22128.28 \u00b1 0.87 kcal/mol,\ncharacterized using HPLC, 1H NMR, and ESI-Ms (Figures respectively. A paired t-test showed significantly lower binding\nS1\u2212S3). Ru-NAP was also synthesized using SPPS. First, energy for Ru-NAP than for NAP (p-value: 0.000091). The\nRu(II)-polypyridyl complex having a pendant carboxy dissociation constant (KD) obtained for Ru-NAP is 1 order of\nfunctionality (Ru-COOH) was synthesized following a magnitude lower than that for NAP suggesting higher affinity\nliterature report with necessary modification in the purification (Ka) binding for Ru-NAP. The respective binding constants of\nprocess (Scheme S2).60\u221263 Cis-dichlorobis(bipyridine)- NAP and Ru-NAP are found to be 9.24 \u00d7 104 and 1.19 \u00d7 106\nruthenium(II) (Ru(2,2\u2032-bipy)2Cl2) and 4\u2032-methyl-2,2\u2032-bipyr- M\u22121 showing higher binding of Ru-NAP compared to NAP\nidine-4-carboxylic acid were dissolved in an ethanol\u2212water (Table S5). Blind docking of NAP and Ru-NAP on the tubulin\n 534 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n\n\n\nFigure 1. Binding interaction of Ru-NAP with tubulin. (a) Tubulin-Ru-NAP complex 1. Snapshots from the MD simulation are superimposed. Ru-\nNAP is shown in green. (b) Close-up view of the last frame of the MD trajectory with interacting residues is highlighted. (c) Tubulin-Ru-NAP\ncomplex 2. (d) Interacting residues. (e) Surface representation shows binding of Ru-NAP (pink) on \u03b2-tubulin (blue). The \u03b1-subunit is shown in\ngreen. Binding constant curves of (f) tubulin and Ru-NAP and (g) tubulin and NAP from the study of quenching of tryptophan fluorescence of\ntubulin in the presence of different concentrations (1\u2212100 \u03bcM) of Ru-NAP or NAP in BRB80 buffer (80 mM PIPES, 1 mM EGTA, 1 mM MgCl2,\npH 6.9) at 25 \u00b0C.\n\ndimers showed potential binding sites including the taxol beta chains are \u221212 each; therefore, the contribution of long-\nbinding sites, as depicted in Figure S20. Further targeted range electrostatic interaction with the cationic Ru(II)-core of\ndocking to the taxol binding site on \u03b2-tubulin produced low- the Ru(II)-polypyridyl moiety significantly drives this\nenergy binding modes (Table S4 and Figure S21). Computa- bimolecular association (Tables S6 and S7).\ntional studies suggest two low-energy binding modes of Ru- Binding of Ru-NAP with Tubulin. The binding of NAP\nNAP: Ru-head is bound to the taxol binding site (Ru-NAP or Ru-NAP to tubulin was evaluated following a conventional\ncomplex 1) and the peptide tail is bound to the taxol binding luminescence quenching process of tryptophan (Trp) of\nsite (Ru-NAP complex 2). Previous literature also suggests the tubulin as a function of [NAP] or [Ru-NAP].69\u221272 Decrease\nbinding of the NAP-peptide to the taxol binding site.68 MD in intrinsic tryptophan fluorescence of tubulin upon binding of\nsimulations showed very stable binding of NAP as well as Ru- NAP or Ru-NAP with tubulin indicates that their binding\nNAP with tubulin throughout the simulation time (Figure causes perturbation of tubulin conformation in the vicinity of\nS22). Figure 1a\u2212e shows a snapshot of MD simulation for the tryptophan residues (Figure 1f, g). The linearity of the Stern\u2212\nRu-NAP and tubulin complexes in two possible binding Volmer plot confirms the dynamic quenching process, while\nconformations. RMSD profiles of the MD simulations, the experimental data show a much higher binding between\ninteracting residues with interaction fractions, and the Ru-NAP and tubulin (KaRu\u2011NAP\u2011tubulin = (6.8 \u00b1 0.55) \u00d7 106\nschematics of the interactions are shown in Figures S23\u2212 M\u22121), as compared to NAP and tubulin (KaNAP\u2011tubulin = (8.2 \u00b1\nS31. Water plays an important role in the binding interaction 1.1) \u00d7 104 M\u22121). This also agrees well with the results of the\nof the peptide, as evident from the several water bridges docking studies. This further corroborates our proposition that\nformed during the MD simulation (Figures S26\u2212S31). It was the cationic Ru(II)-polypyridyl moiety in Ru-NAP favors a\nalso observed that the bipyridyl moieties of the Ru-NAP stronger binding to \u03b2-tubulin than NAP.\nheadgroup may interact with the tyrosine side chains by Ru-NAP Forms Fibrillar Aggregates. NAP peptide is\nforming pi-stacking interactions (Figures 1a\u2212e and S31). The known to form fibrillar structures when incubated for 7\nrole of the cationic Ru(II)-polypyridyl moiety is important in days.73,74 We studied the morphology of NAP, Py-NAP, and\nthe binding of Ru-NAP with tubulin as tubulin remains highly Ru-NAP after incubation for 5 days in aq 1\u00d7 PBS buffer using\nnegatively charged at neutral pH. Charges on tubulin alpha and transmission electron microscopy (TEM). NAP forms the\n 535 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\nfibrillar structure as previously reported (Figure 2a,b). TEM which is found to be improved in the case of Py-NAP and Ru-\nimages recorded for Py-NAP also showed fiber-like morphol- NAP (Figure 2f,g). Figure S32 shows the histogram of the\n average diameter of the NAP, Py-NAP, and Ru-NAP fibers.\n These indicate that Rubpy favored the self-assembly process in\n Ru-NAP.\n Dark Stability and Photostability of Ru-NAP. We\n investigated the dark and photostability of the Ru-NAP and\n compared these with Rubpy. Both complexes showed\n insignificant degradation in the dark when incubated in 1\u00d7\n PBS buffer (pH 7.4) for 12 h (Figures 2h and S33). The\n stability of Rubpy and Ru-NAP after successive irradiation\n with a 450 nm laser (200 mW\u00b7cm\u22122, 1 min, 12 J\u00b7cm\u22122) in 1\u00d7\n PBS buffer (pH 7.4) was examined (ESI). Only insignificant\n bleaching was observed for Rubpy, while a slight degradation\n (<7%) was observed for Ru-NAP due to photobleaching even\n after 5 cycles (Figures 2i and S33). This ensured that the\n conjugation of NAP to Rubpy in Ru-NAP had no influence on\n its dark stability, while a marginal decrease in its photostability\n characteristics was observed.\n Type II ROS Generation of Ru-NAP. To investigate the\n efficacy of Ru-NAP in generating 1O2, we performed the 1,3-\n diphenylisobenzofuran (DPBF) assay in Milli-Q water\n containing 1% DMSO (v/v). The absorbance of DPBF was\n found to decrease gradually when DPBF was irradiated for 15 s\n in the presence of Ru-NAP with a 450 nm laser (Figures 3a\n and S34a,b). However, the absorbance of DPBF remained\n unaltered when identical experiments were performed without\n any 450 nm irradiation. This confirmed the in situ ROS\n generation on irradiation of Ru-NAP at 450 nm. The DPBF\nFigure 2. (a) Schematic representation of the self-assembly of NAP, assay is used for the detection of 1O2, apart from other ROS\nPy-NAP and Ru-NAP. TEM images of (b) NAP (100 \u03bcM; 1\u00d7 PBS species.76 To reconfirm the in situ generation of 1O2 as a\nbuffer (pH = 7.4), while (e) represents the corresponding SEM transient species, we further performed an additional 9,10-\nimage. (c) TEM image of Py-NAP (100 \u03bcM; 1\u00d7 PBS buffer (pH = anthracenediyl-bis(methylene)dimalonic acid (ABDA) assay,\n7.4) containing 2.5% DMSO (v/v), while (f) represents the\ncorresponding SEM image. (d) TEM image of Ru-NAP (100 \u03bcM; which is specific for 1O2.77 A successive decrease in the\n1\u00d7 PBS buffer (pH = 7.4) containing 2.5% DMSO (v/v), while (g) absorbance of ABDA was observed after irradiation with 450\nrepresents the corresponding SEM image. (h) Dark stability of Rubpy nm light in the presence of Ru-NAP in Milli-Q water. The\nand Ru-NAP using 75 \u03bcM of each of these complexes in 1\u00d7 PBS reaction of ABDA with 1O2, generated in situ, on irradiation of\nbuffer (pH = 7.4) containing 1% DMSO (v/v). Absorbances were Ru-NAP, accounted for this observed gradual decrease in\nrecorded in 3 h intervals up to 12 h. (i) Photo stability of Rubpy and absorbance (Figures 3b and S34c,d). However, control\nRu-NAP using 75 \u03bcM of each of these complexes in 1\u00d7 PBS buffer experiments revealed that the absorbance of ABDA remained\n(pH = 7.4) containing 1% DMSO (v/v). Absorbance was recorded unaltered for Ru-NAP treated solution without photo\nafter irradiation for 1 min with 450 nm laser light (200 mW\u00b7cm\u22122; 1\nmin, 12 J\u00b7cm\u22122) in 15 min intervals up to 75 min. The \u00b1SD was irradiation. This further corroborated our presumption that\ncalculated from three independent sets of experiments. Ru-NAP belonged to the Type II PDT agent. The DPBF and\n ABDA assays for Rubpy also confirm 1O2 generation but to a\nogy (Figure 2c). Better fiber formation for Py-NAP, compared lesser extent than Ru-NAP (Figure S35). The quantum yield\nto NAP under identical conditions, could be ascribed to the for 1O2 generation (\u03941O2) for Ru-NAP was evaluated as 0.55\nability of the Py moiety to participate in cation\u2212\u03c0/\u03c0\u2212\u03c0 by UV\u2212vis titration studies of DPBF using methylene blue\nstacking interactions. Importantly, the degree of fiber (MB) as a reference (Figure S36). This is a reasonably high\nformation was more extensive for Ru-NAP, as compared to value because we used Milli-Q water medium saturated with\nNAP or Py-NAP (Figure 2d). Literature reports suggest that O2 and is higher than the desired value (\u03941O2 = 0.5) for any\nthe cationic FeII(2,2\u2032-bipy)32+ moiety in FeII(2,2\u2032-bipy)32+- PDT applications.78 These data confirmed the therapeutic\ncollagen conjugate favors the self-assembly process and fiber potential of Ru-NAP on irradiation with 450 nm light. Next,\nformation.75 A similar phenomenon was observed in the the possibility of the generation of Type I ROS (hydroxyl\npresent study. Importantly, the extent of fiber formation for radical (\u2022OH) or/and superoxide radical (O2\u2022\u2212)) as a transient\nPy-NAP was lower than that of Ru-NAP when both were species on irradiation of Ru-NAP with 450 nm light was\nsubjected to self-assembly to form fibers under identical examined. Typical terephthalic acid (TPA) and 5,5\u2032-dimethyl-\nconditions. This presumably arises from a head-to-tail 1-pyrroline N-oxide (DMPO)-based assays were performed\norientation of the Ru-NAP molecules during the self-assembly respectively to identify the formation of transient ROS species\nprocess for fiber formation (Figure 2a). The self-assembly like \u2022OH and O2\u2022\u2212 on irradiation of Ru-NAP with 450 nm\nbehavior of NAP, Py-NAP, and Ru-NAP was further light (Figure S37, ESI). These results confirm that Ru-NAP\nconfirmed using scanning electron microscopy (SEM). The failed to induce the formation of Type I PDT under the\nSEM images for NAP show short fiber formation (Figure 2e), experimental conditions.79,80\n 536 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n\n\n\nFigure 3. (a) Changes in absorbance of DPBF treated with Ru-NAP ([DPBF] = 125 \u03bcM and [Ru-NAP] = 2.5 \u03bcM in Milli-Q water containing 1%\nDMSO (v/v)) were recorded at 419 nm following irradiation with 450 nm light (200 mW\u00b7cm\u22122, 15 s, 3 J\u00b7cm\u22122). (b) Changes in absorbance of\n[ABDA] (80 \u03bcM) treated with [Ru-NAP] (1.6 \u03bcM) in Milli-Q water under irradiation of 450 nm light (200 mW\u00b7cm\u22122, 1 min, 12 J\u00b7cm\u22122). (c)\nDetermination of lipophilicity of NAP, Rubpy and Ru-NAP by the shake-flask method using an equal mixture of an octanol\u2212water mixture. The\nlipophilicity of Ru-NAP was found to be increased compared to the Rubpy. (d) Fluorescence-based tubulin polymerization assay of NAP and Ru-\nNAP using the fluorescence-based tubulin polymerization assay (Cat. # BK011P) from Cytoskeleton. (e) Stability of Ru-NAP (%) and (f) time-\ndependent HPLC of Ru-NAP in fetal bovine serum (FBS, 20% v/v in 1\u00d7 PBS buffer (pH = 7.4)) up to 24 h. The HPLC analysis of the Ru-NAP\n(100 \u03bcM) incubated sample in 20% FBS showed >80% stability up to 24 h.\n\n\n\n\nFigure 4. Representative CLSFM images showing accumulation of Ru-NAP (Ru-NAP shows intrinsic green fluorescence) in live MDA-MB-231\ncells (a) incubated with Ru-NAP (5 \u03bcM for 4 h; scale bar, 5 \u03bcm) at 37 \u00b0C, (b) untreated (upper row) and incubated with 5 \u03bcM Ru-NAP for 4 h at\n37 \u00b0C (middle row) or 4 \u00b0C (bottom row), and (c) preincubated with 400 mM sucrose for 30 min (upper row) or 10 mM M-\u03b2-CD for 30 min\n(middle row) or 3 mM amiloride for 15 min (bottom row).\n\n Lipophilicity Measurement of Ru-NAP. Appropriate internalization. Lipophilicity takes care of the solvation ability\nlipophilic balance rather than the overall charge of the of molecules in fats and lipids as well as in aqueous media. It\ncompound is an important parameter for effective cellular can be correlated with cellular internalization and up-\n 537 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n\n\n\nFigure 5. (a) Representative CLSFM images showing colocalization of Ru-NAP (20 \u03bcM for 4 h; intrinsic green fluorescence) with MT (red\nfluorescence). Co-localization is shown in the merged image. Merged image indicates the nice colocalization of Ru-NAP with the MT (Pearson\u2019s\ncoefficient: 0.88). Dose\u2212response profiles of (b) Ru-NAP under dark and after light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) and (c)\nRubpy under dark and after light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) from the toxicity data using the MTT assay. (d) Hill plot\nof the toxicity data of the Ru-NAP followed light treatment. (e) Cell cytotoxicity of the Ru-NAP and Rubpy was measured using the MTT assay\nafter light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) in HEK293 cells. (f) Dark toxicity of Ru-NAP and Rubpy in HEK293 cells.\n\ntake.54,81,82 The distribution coefficients (log P) of NAP, \u03bcM) incubated sample in both PBS and 20% FBS showed >95\nRubpy, and Ru-NAP were evaluated in an octanol\u2212water and >80% stability, respectively, until 24 h due to the small size\nmixture using the shake-flask method. The evaluated lip- and the N-stapled termini of the NAP peptide (Figures 3e,f\nophilicity of Ru-NAP was higher than that of Rubpy (Figures and S39).\n3c and S38). Higher lipophilicity improves the cellular Cellular Uptake of Ru-NAP. NAP is known to be a cell-\ninternalization process and helps to overcome accumulation penetrating peptide. As discussed earlier, presumably, the\ndefects with consequential cytotoxicity.83,84 endocytosis pathway rather than membrane translocation\n Effect of Ru-NAP on Tubulin Dynamics, Buffer, and prevailed at the low concentration used for our studies at\nSerum Stability of Ru-NAP. A literature report suggests that physiological conditions.86 We checked the morphology of Ru-\nNAP interacts with MT end-binding proteins (EB1 and EB3) NAP in an RPMI medium supplemented with 10% fetal bovine\nand increases the dendritic spine density.85 We examined the serum used for the cell culture. The TEM and SEM images of\nbinding of the Ru-NAP to the tubulin and its\u2019 influence on the Ru-NAP also show the fiber formation (Figure S40). Next, we\ntubulin polymerization process using a fluorescence-based investigated the effective cellular uptake of Ru-NAP using\ntubulin polymerization assay (BK006P). The extent of the light high-resolution live cell confocal laser scanning fluorescence\nscattered by MT is proportional to the concentration of the microscopy (CLSFM) imaging in the MDA-MB-231 cell. The\nmicrotubule polymer. A plot of the extent of light scattered as a MDA-MB-231 cell line is ER, PR, and E-cadherin negative and\nfunction of the time yielded the polymerization curve depicting expresses mutated p53. These cells also lack the growth factor\nthe nucleation, growth, and steady-state equilibrium (Figure receptor HER2 and represent a good model of triple-negative\n3d). It is evident from Figure 3d that Ru-NAP is more efficient breast cancer.87 Four hours post-treatment was found to be\nin stabilizing the microtubule and enhances tubulin polymer- adequate for the internalization of Ru-NAP (5 \u03bcM) into the\nization in comparison to NAP (used in equimolar concen- cellular cytoplasm (Figure 4a; see merged image with bright\ntration), which was used as a positive control. Next, we field). We further checked the cellular uptake of Ru-NAP in\nchecked the stability of Ru-NAP in 1\u00d7 PBS buffer (pH = 7.4) MDA-MB-231 and HEK293 cells using inductively coupled\nand fetal bovine serum (FBS, 20% v/v in 1\u00d7 PBS buffer (pH = plasma-mass spectrometry (ICP-MS). The result shows a\n7.4)) up to 24 h. The HPLC analysis of the Ru-NAP (100 \u223c2.6-fold higher accumulation of Ru-NAP in live MDA-MB-\n 538 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n231 cells compared to that of live HEK293 cells (Figure S41). binding to many MT-binding proteins, which collectively have\nWe have performed new live cell CLSFM by incubating live a serious implication on cellular motility and cell death.56,89\nMDA-MB-231 cells with Ru-NAP at 37 and 4 \u00b0C to examine Therefore, we assessed the dark toxicity (chemotherapeutic\nactive or passive uptake of the drug. Interestingly, we observed effect) of the Ru-NAP in MDA-MB-231 cells for 24 h using\na 90% reduction in Ru-NAP Mean Fluorescence Intensity the MTT assay. Ru-NAP shows certain cytotoxicity in the dark\n(MFI) inside cells at 4 \u00b0C compared to 37 \u00b0C suggesting an and the corresponding IC50 value is 2.9 \u00b1 0.5 \u03bcM indicating\nenergy-dependent uptake of Ru-NAP in live MDA-MB-231 the significant chemotherapeutic effect of the Ru-NAP due to\ncells through the endocytosis process (Figures 4b and S42). the binding of the Ru-NAP fibers to the tubulin/MT of the\nThis indicates the active cellular internalization of Ru-NAP. To MDA-MB-231 cells (Figure 5b). The dark toxicity of the Ru-\nfurther confirm the role of endocytosis for the Ru-NAP uptake NAP is much higher than that of Rubpy (IC50 of Rubpy under\ninside cells, we have applied a range of endocytosis inhibitors, dark is 21.1 \u00b1 4.2 \u03bcM; Figure 5c). Next, we assessed the PDT\nnamely, sucrose (Clathrin endocytic inhibitor), methyl-\u03b2- efficacy of the Ru-NAP and compared the same with the\nCyclodextrin (M-\u03b2-CD; caveolin endocytic inhibitor), and Rubpy as the control. The MDA-MB-231 cells were incubated\namiloride (macropinocytosis inhibitor). CLSFM data indicate with varying concentrations of Ru-NAP and Rubpy for 4 h\na significant reduction in Ru-NAP uptake (p < 0.001) in cells followed by light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min,\nfollowing pretreatment with sucrose (Figures 4c and S42). 60 J\u00b7cm\u22122) and further kept in the dark for another 24 h. The\nMDA-MB-231 cells pretreated with M-\u03b2-CD (caveolin cytotoxicity data revealed that Ru-NAP showed significant\nendocytic inhibitor) also show a 5-fold reduction in cellular light toxicity to the MDA-MB-231 cells with a LightIC50Ru\u2011NAP\nuptake of Ru-NAP. However, pretreatment with amiloride value of 32.5 \u00b1 7.8 nM, a value that is \u223c89 times lower than\nshowed insignificant changes in Ru-NAP cellular uptake under Ru-NAP (DarkIC50Ru\u2011NAP = 2.9 \u00b1 0.5 \u03bcM, Figure 5b) due to its\nsimilar conditions. These results indicate that clathrin- and combined effect of chemotherapeutic through the induction of\ncaveolin-dependent endocytic processes are involved in the higher MT polymerization and PDT. It is noteworthy that\nuptake of Ru-NAP in MDA-MB-231 cells. To ascertain the Rubpy shows certain cytotoxicity in the dark and upon light\nmolecular aggregate inside the live MDA-MB-231 cells, we irradiation. The IC50 of dark toxicity of Rubpy [DarkIC50Rubpy] is\nfurther used fluorescence imaging studies using NBD-NAP. 21.1 \u00b1 4.24 \u03bcM and the IC50 of light toxicity of Rubpy\nBeing an environmentally sensitive fluorophore, NBD is Light\n IC50Rubpy (0.24 \u00b1 0.05 \u03bcM; Figure 5c). Most importantly,\nfrequently used to ascertain the formation of molecular Dark\n IC50Ru\u2011NAP (2.9 \u03bcM) is lower by \u223c7.2 times and Light-\nassembly(ies) in the cellular milieu. NBD derivatives typically\n IC50Ru\u2011NAP value is lower by \u223c7.3 times than that for\nshow strong fluorescence as they get readily partitioned into Dark\n IC 50 Rubpy (21.1 \u03bcM) and LightIC 50 Rubpy (0.24 \u03bcM)\nhydrophobic pockets and this is accompanied by a character-\nistic green NBD fluorescence maximum (\u03bbEm = 540 nm) respectively. Thus, these data reveal that Ru-NAP is more\nfollowing excitation at 470 nm.88 Internalization of NBD-NAP cytotoxic than Rubpy due to its\u2019 chemotherapeutic effect and\nwith subsequent partitioning within the fibrillar aggregates in Ru-NAP on light irradiation shows a substantially low IC50\nlive MDA-MB-231 showed a strong green fluorescence to value of 32.5 nM toward live MDA-MB-231 cancer cells\nconfirm the significant fiber of NBD-NAP (Figure S43). (Figure 5b,c, Table S8).55 The NAP peptide alone did not\nHowever, such a fiber is less prominent in HEK293 cells due to show light or dark toxicity toward live MDA-MB-231 cells up\nless cellular uptake compared to MDA-MB-231 cells (Figure to 200 \u03bcM concentration and this agreed well with the\nS44). This supports our presumption of intracellular fibers of literature reports (Figure S46).36 To check the PDT effect of\nanalogous molecules of Ru-NAP. Ru-NAP in nontumorigenic normal human embryo kidney\n Co-Localization of Ru-NAP with Tubulin/MT. To verify cells (HEK293) cells, these cells were incubated with varying\nif Ru-NAP was localized on the MTs, we incubated MDA-MB- concentrations of Ru-NAP for 4 h in the dark. These\n231 cells with Ru-NAP (20 \u03bcM for 4 h), costained them with pretreated cells were then irradiated for 5 min (450 nm, 200\nthe microtubule-specific marker (EP1332Y), and analyzed mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) and kept in the dark for another\nthem via CLSFM (Figure 5a). Significant colocalization of Ru- 24 h. It showed a much lower toxicity toward HEK293 cells\nNAP with tubulin was detected, suggesting a strong association ( LightIC50Ru\u2011NAP [HEK293] 17.2 \u00b1 2.5 \u03bcM; Figure 5e)\nof Ru-NAP with MTs. A detailed plot profile analysis compared to MDA-MB-231 cells (LightIC50Ru\u2011NAP value of\ndemonstrates significant colocalization of the green-colored 32.5 nM). Importantly, Ru-NAP under dark incubation for 24\nRu-NAP and red-colored tubulin (Figure S45). Co-localization h did not show detectable toxicity up to 80 \u03bcM toward\nanalysis of Ru-NAP (green) with tubulin (red) showed a HEK293 cells (Figure 5f). However, Rubpy shows significant\nPearson Coefficient (R) of 0.88. This seems rational dark and light toxicity to the HEK293 cells (Figure 5e,f). The\nconsidering the improved lipophilicity and higher surface NAP peptide alone did not show light or dark toxicity up to\ncharge affinity for Ru-NAP as compared to NAP, which is 200 \u03bcM concentration toward live HEK293 cells (Figure S47).\notherwise known to bind preferentially to \u03b2-tubulin. These We further checked the phototoxicity of the Ru-NAP after\ncontributed to the higher evaluated binding constants for Ru- irradiation with green light (520 nm) and red light (630 nm),\nNAP compared to NAP (control) toward \u03b2-tubulin and apart from the blue light (450 nm). Irradiation with red light\nconfirmed the high targeting ability of the Ru-NAP to MT in shows insignificant phototoxicity (IC50 is similar to that of dark\nlive MDA-MB-231 cells. toxicity), while this is most prominent when blue light (450\n Cytotoxicity Study of Ru-NAP. MT serves as \u201cfreeways\u201d nm) is used (Figure S48b and Table S8). Irradiation with\nfor intracellular trafficking. Such fiber formation on the MT green light shows a slightly lower PDT effect compared to the\nsurface and/or inside the tubular structure of MT would one observed with blue light (Figure S48a and Table S8). The\nfurther interfere with the intracellular trafficking of vesicles and minimal PDT effect of red light compared to the green light\norganelles, transport of cargo by motor proteins from the and blue light correlates well with the relative difference in the\nkinesin/dynein superfamilies and ion transport, apart from molar absorptivity of Ru-NAP in the respective spectral zone.\n 539 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n\n\n\nFigure 6. (a) Effect of Ru-NAP on the MT/tubulin network in live MDA-MB-231 was studied using CLSFM. A significant disruption of the MT\nnetwork of the live MDA-MB-231 cells was evident for Ru-NAP treatment both under dark and with light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5\nmin, 60 J\u00b7cm\u22122). (b) Effect of Ru-NAP on the Actin network (stained using Alexa Fluor 568 Phalloidin) in live MDA-MB-231 was studied in\nCLSFM. There is no change in the Actin network of the live MDA-MB-231 cells for Ru-NAP (20 \u03bcM, 4 h) treatment. (c) Representative Western\nblots for tubulin, actin, and GAPDH in MDA-MB-231 cells; either untreated or treated with Ru-NAP (5 or 20 \u03bcM) for 4 h with or without blue\nlight photoactivation. GAPDH is shown as the loading control. The migration of protein molecular weight markers is indicated on the right. (d)\nDensitometry analysis represents the fold change in tubulin, and actin, normalized to GAPDH (error bars represent means \u00b1 SEM). Asterisks\ndenote statistically significant differences (***P < 0.001, t test).\n\nWe further studied the dark toxicity and phototoxicity of the 2-deoxy-D-Glucose (inhibitor of glycolysis), allowing us to\nRu-NAP under blue light irradiation in human cervical cancer investigate the Warburg effect. The Mean Fluorescence\n(HeLa) cells and human breast cancer (MCF7) cells. Ru-NAP Intensity (MFI) of Ru-NAP decreased \u223c5 fold in the presence\nshowed similar dark and phototoxicity to that of MDA-MB- 2-deoxy-D-glucose which indicates a 5-fold reduction in the\n231 cells (Figure S49). Table S8 reveals the IC50 value of Ru- drug uptake into cancer cells in the presence of glycolysis\nNAP and Rubpy in different cancer and normal cells, along inhibitors and provides direct evidence for the operation of the\nwith their respective phototoxic index (PI; IC50 under dark/ Warburg-phenomenon-dependent Ru-NAP cellular uptake\nIC50 under light irradiation). The observed high toxicity of Ru- (Figure S50). Thus, the above results reveal that the\nNAP in MDA-MB-231 cells compared to HEK293 cells conjugation of NAP to Rubpy has a profound effect in\npresumably lies in the accelerated metabolic rate of MDA-MB- reducing the IC50 of MDA-MB-231 cells compared to two\n231 cells as a result of the Warburg effect and the tendency of individual candidates (NAP and Rubpy). These indicate the\nsmall molecules to accumulate in cells once they have formed potentiality of Ru-NAP as a bimodal PDT agent compared to\naggregates as in the case of Ru-NAP.90\u221292 We utilized live-cell Rubpy due to the microtubule targeting ability, fiber\nhigh-resolution CLSFM to test the uptake of Ru-NAP (5 \u03bcM formation, and PDT effect. It is worth mentioning here the\nfor 4 h) in MDA-MB-231 cells in the ATP-depleted M1 IC50Light of various derivatives (with small organic molecules)\nmedium supplemented with 10 mM sodium azide and 10 mM of Ru(II)-polypyridyl complexes toward various live cancer\n 540 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\ncells are typically >5 \u03bcM and considering these, the observed Cell Cycle Analysis. We examined the cell cycle of MDA-\nIC50Light (32.5 nM) of Ru-NAP is significant.27,55,93 The cell MB-231 cells using PI/RNase staining following incubation\nviability vs log[Ru-NAP] under light irradiation plot, a dose\u2212 with Rubpy or Ru-NAP (each of 20 \u03bcM for 4 h) and\nresponse plot, is sigmoidal (Figure 5b) in nature. It has been photoactivation followed by light irradiation (450 nm, 200\nestablished that the sigmoidal plots are better than linear ones mW\u00b7cm\u22122, 5 min) and incubation for another 48 h. Treatment\nfor explaining the dose-dependent drug efficacy94 as this with Rubpy + L or Ru-NAP + L induced a noticeable change\ndemonstrates the \u03b2-tubulin-specific Ru-NAP induces a in the cell cycle profile (Figure 7a). Results for the Rubpy + L\ncooperative influence as obtained from the Hill plot (n =\n treatment at 20 \u03bcM revealed a G2/M phase arrest (12.2%)\n1.14) in terms of cellular killing by 1O2 and chemo toxic effect\nof Ru-NAP fibers (Figure 5d). n > 1 in the Hill plot indicates compared with the untreated control (11.3%). In contrast, the\nthe cooperative influence. Next, we performed the dark and use of 20 \u03bcM Ru-NAP + L showed enhanced G2/M phase\nphototoxicity studies of Ru-NAP under hypoxia (1% O2) to arrest (24.3%). Docking studies revealed that Ru-NAP was\nconfirm the role of MT targeting in the management of tumor bound to the \u03b2-tubulin part of the MT more efficiently than\nhypoxia. It shows similar toxicity as in normoxia (21% O2),\nwhich indicates that the MT targeting is beneficial for Ru-NAP\nas a potential Type II PDT agent (Figure S51). Table S8\nindicates the corresponding IC50 and PI of Ru-NAP in hypoxia\nand normoxia.\n Immunocytochemistry for the Investigation the\nEffect of Ru-NAP on the MT/Tubulin Network. The\nhigh targeting ability of the MTs and the significant PDT\nefficacy of Ru-NAP motivated us to examine the mechanistic\npathway for cell death. MTs comprise \u03b1 and \u03b2-tubulin\nheterodimers that undergo dynamic cycles of polymerization\nand depolymerization processes. A dynamic balance ensures\nproper cellular functions and survival.12 To test the dynamics\nof the MT network following the treatment with Ru-NAP and\nthen photoactivation, we performed immunocytochemistry\nwith anti-tubulin antibody in live MDA-MB-231 cells exposed\nto Ru-NAP. Without photoactivation, Ru-NAP treatment (20\n\u03bcM for 4 h) had a significant impact on the MT network,\nindicating perturbation in structural integrity due to the\nbinding of the Ru-NAP fibers to MT, which accounts for its\nchemotherapeutic effect under dark (Figure 6a). Moreover,\nanalogous experiments with light activation showed a more\nsignificant dose-dependent disruption of the MT network,\nsuggesting an imbalance in the polymerization-depolymeriza-\ntion cycle. This agreed well with the fluorescence-based tubulin\npolymerization assay showing enhanced tubulin polymer-\nization in the presence of Ru-NAP. As a part of the detailed\nmechanism studies, we checked the effect of Ru-NAP on the\nActin network of MDA-MB-231 cells. There is no change in\nthe actin network of the live MDA-MB-231 cells treated with\n20 \u03bcM Ru-NAP (Figure 6b). Moreover, there is no\ncolocalization of the Ru-NAP with the actin network,\nindicating the precise targeting of the Ru-NAP to the\ntubulin/MT (Figure 6b). To further validate the tubulin-\nspecific binding/activity of Ru-NAP, which promotes specific\ndegradation of the tubulin network in cells, we performed a Figure 7. (a) Histograms of the distribution of DNA content by flow\ntubulin degradation assay following Ru-NAP treatment using cytometric analyses of the Ru-NAP mediated cell cycle arrest. (b)\nWestern blotting. We have tested the expression pattern of the Flow cytometry analysis of cellular ROS using CM-H2DCFDA in\nprincipal cytoskeletal component (tubulin and actin with MDA-MB231 cells, either untreated or treated with Rubpy or Ru-\nGAPDH) after Ru-NAP treatment, coupled with photo- NAP (20 \u03bcM) separately for 4 h followed by photoactivation (450\nactivation. We detected a significant (4-fold) reduction in nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122). (c) Analysis of the apoptosis in\nthe tubulin levels compared to Actin after the cells were treated flow cytometry using dead cell apoptosis kit with Annexin V Alexa\nwith Ru-NAP (Figure 6c,d). Under similar conditions, both Fluor 488 and propidium iodide (PI) (catalogue number: V13241)\nActin and GAPDH levels remained unchanged (see the using Dox (5 \u03bcM) as a positive control. Ru-NAP induces apoptosis in\n the MDA-MB-231 cells under dark conditions and after light\nquantification). These results provide direct evidence for the\n irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122). (d) Histogram\ntubulin-specific binding and degradation by Ru-NAP without of percentages of healthy cells, apoptotic cells, and necrotic cells after\nperturbation of the actin protein, and the cytotoxicity of the different treatments (i: untreated, ii: Dox, iii: Rubpy + L, iv: Ru-NAP,\nRu-NAP is a result of the combined effect of tubulin/MT v: Ru-NAP + L) in MDA-MB-231 cells, measured using flow\ndisruption and huge site-specific 1O2 generation\ufffda distinct cytometry. The data represent the mean value \u00b1 SD obtained from\ndemonstration of CALI. three independent experiments.\n\n 541 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\nNAP and, presumably, this had a pronounced influence in irradiation for 5 min (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7\nstabilizing tubulin polymerization. This was also consistent cm\u22122). This was repeated every alternative day for 3 weeks.\nwith colocalization studies and in vitro tubulin polymerization The tumor sizes were measured using a slide caliper, and the\nexperiments, which confirmed that Ru-NAP could stimulate tumor volume was calculated using the formula 0.5 x length x\nMT polymerization to disrupt MT dynamics. Seemingly, this width2 mm3. The tumor size and the mice's body weight were\ninterferes with the mitotic spindle assembly during the recorded every third day for 3 weeks. On the 21st day, each\nmetaphase/anaphase transition, triggers the mitotic spindle mouse was sacrificed, the tumor was isolated and the weight of\nassembly checkpoint, and causes G2/M arrest. Our results are each tumor was recorded. A significant decrease in the tumor\nsimilar to the classical anticancer drugs like Paclitaxel, which sizes was observed in the Ru-NAP+ L treated group compared\ninduces cell cycle arrest by stabilizing MTs and triggering to the control and Rubpy + L (Figure 8a). From the tumor\napoptosis.95\n Intracellular ROS Generation. Next, we performed flow-\ncytometry-based analysis to quantify ROS produced in situ due\nto photoirradiation of live MDA-MB-231 cells after treatment\nwith Rubpy or Ru-NAP or hydrogen peroxide (H2O2) as a\npositive control using CM-H2DCFDA. We detected significant\nROS production upon exposure to H2O2 (500 \u03bcM, 12 h;\nFigure S52). Moreover, a significant increase in intracellular\nROS for Ru-NAP + L (20 \u03bcM) treated cells was observed\ncompared to Rubpy + L (Figure 7b). These results are\nconsistent with data showing that Ru-NAP is a Type II PDT\ninducer that generates 1O2 (vide infra) with a relatively high\nquantum yield of 0.55. These confirmed that the cytotoxicity of\nRu-NAP was a combination of ROS-induced cell death and\nsignificant disruption of MT dynamics. Taken together, Ru-\nNAP destabilized MTs which triggered cell cycle arrest at the\nG2/M phase along with ROS-induced cell death, which\naccounted for the observed cellular cytotoxicity (LightIC50Ru\u2011NAP\n= 32.5 \u00b1 7.8 nM) in MDA-MB-231 cancer cells.\n Flow Cytometry for Analysis of the Apoptosis\nPathway. The mode of cell death is an important parameter\nfor any chemotherapeutics drugs. We investigated the cell\ndeath pathway induced by Ru-NAP by flow cytometry using\nFITC-Annexin V and Propidium Iodide using Doxorubicin\n(Dox) as a positive control for the induction of apoptosis.96,97 Figure 8. Tumor growth inhibition study of Ru-NAP followed by\nFITC(\u2212)PI(\u2212) quadrant indicates healthy cells, FITC(+)- light irradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) using\nPI(\u2212) and FITC(+)PI(+) indicate the apoptotic cells, and MDA-MB-231 tumor xenograft model in balb/c nude mice. (a) The\nFITC(\u2212)PI(+) indicates the necrotic cells. The cells were images of the tumor of different group (n = 5) isolated from the mice\ntreated with Ru-NAP (1 \u03bcM) for 4 h followed by light at 21st day after the treatment. The result showed a significant\nirradiation (450 nm, 200 mW\u00b7cm\u22122, 5 min, 60 J\u00b7cm\u22122) and decrease in the tumor sizes of the Ru-NAP + L treated group\nkept in the dark for another 20 h. The cells were analyzed in compared to untreated and Rubpy + L. (b) Tumor growth ratio curve\n of the different treatment group (n = 5). The growth ratio for the Ru-\nflow cytometry following incubation with annexin V and PI. A NAP+ L treated group is very low compared to those of the untreated\nsignificant fraction (97%) of MDA-MB-231 cells underwent and Rubpy + L. (c) Histogram of the average tumor weight of the\nthe apoptosis process for Ru-NAP treated after light irradiation different treatment group (n = 5) as indicated. Tumor weight is too\n(Figure 7c); 44, 38, and 47% of cells underwent apoptosis after low for Ru-NAP + L with respect to that of untreated and Rubpy + L.\nRubpy + L, Ru-NAP, and Dox treatment, respectively (Figure A significant difference was analyzed by comparing with the\n7c). Figure 7d shows the histogram of quantitative analysis nonsample-treated control group. * represents P < 0.05, ** represents\nfrom the triplicate experiment. This indicated that Ru-NAP P < 0.01, and *** represents P < 0.001. The P value was obtained\nshowed significant apoptosis under dark conditions and almost using student\u2019s t test. (d) Change in the mice bodyweight of the\nexclusive light-induced apoptosis of the MDA-MB-231 cells different treatment group (n = 5) monitored for 3 weeks. (e) H&E\n staining of the major organs (kidney, heart, liver, lung, and spleen)\nthrough a combination of disruption of the microtubule/\n after Ru-NAP treatment in nude mice showing no abnormalities,\ntubulin network dynamics and ROS generation and the efficacy indicating that Ru-NAP is not toxic to the mice.\nof Ru-NAP was more pronounced than Rubpy.\n In Vivo Studies of Ru-NAP. The PDT efficacy of Ru-NAP\nwas further verified in the in vivo MDA-MB-231 xenograft growth ratio curve, 90% and 60% inhibition of the tumor\nmodel in nude mice. The MDA-MB-231 cells were inoculated growth was observed in 3 weeks for Ru-NAP + L- and Rubpy\nin the right flank of the mice. When the tumor size reached + L-treated groups, respectively, compared to the control\n\u223c100 mm3, the mice were divided into three groups with n = (Figure 8b). There was a 90.5 and 56.6% reduction in the\n5. The groups are (i) untreated, (ii) Rubpy + L-treated, and tumor weight for the Ru-NAP + L- and Rubpy + L-treated\n(iii) Ru-NAP + L-treated. The mice with an abnormal size group, respectively, compared to the control (Figure 8c). We\nwere excluded from the study. Then, the mice were treated further checked the tumor growth inhibition of Ru-NAP (2\nwith Rubpy or Ru-NAP (2 mg/kg per mouse) intravenously mg/kg per mouse) under dark conditions using the MDA-MB-\nand then kept in the dark for the next 24 h followed by light 231 xenograft model in nude mice with n = 5 to check the\n 542 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\nchemotherapeutic effect of the Ru-NAP. The tumor images inducing cell death. Ru-NAP also shows highly effective tumor\nand tumor growth ratio curve show 50% inhibition of the growth inhibition in the MDA-MB-231 tumor xenograft model\ntumor growth in 3 weeks after Ru-NAP treatment (Figure in nude mice. Through this report, we can establish that a new\nS53). These data confirmed that Ru-NAP could induce tumor Ru-NAP is used effectively for treating TNBC without\ngrowth inhibition under the dark through a chemotherapeutic inducing systemic toxicity.\neffect and a more effective growth inhibition through MT\ndisruption, as well as PDT-induced toxicity by 1O2. There was\nno change in the body weight of the mice after Ru-NAP + L\n \u25a0\n *\n ASSOCIATED CONTENT\n s\u0131 Supporting Information\nand Rubpy + L treatment compared to the control (Figure\n8d). Moreover, the H&E staining data of the major organs The Supporting Information is available free of charge at\nafter Ru-NAP treatment do not show any abnormalities https://pubs.acs.org/doi/10.1021/jacs.4c11820.\n(Figure 8e). This indicates that Ru-NAP was nontoxic to the Experimental section; peptide synthesis; Ru-NAP syn-\nmice. The in vivo study further confirmed that Ru-NAP thesis; characterization; lifetime decay; docking and\npossessed a significant tumor inhibition effect with insignificant simulations; tryptophan quenching; DPBF and ABDA\nin vivo toxicity. assay; lipophilicity; serum stability, morphology in\n cellular media; cellular uptake and endocytosis; ICP-\n\u25a0 CONCLUSIONS\nIn conclusion, we have demonstrated a proof-of-concept in\n MS; tubulin colocalization, hill plot, immunocytochem-\n istry, Western blot, cell cycle; DCFDA; annexin V/PI;\n and in vivo studies (PDF)\ndeveloping an efficient PDT agent (Ru-NAP) for treating\nTNBC by conjugating a photoactive Ru(II)-polypyridyl\nmoiety, efficient in generating in situ 1O2, to serum stable\noctapeptide, NAP. NAP with N-stapled termini and being a\n \u25a0 AUTHOR INFORMATION\n Corresponding Authors\nshort peptide presumably evades proteolytic degradation in\nblood serum. The TEM images of the Ru-NAP/Py-NAP/ Anitha Ethirajan \u2212 Institute for Materials Research (Imo-\nNAP, incubated for 5 days, reveal that the fiber formation was imomec), Nanobiophysics and Soft Matter Interfaces (NSI)\nmost extensive for Ru-NAP, which is attributed to a favored Group, Hasselt University, B-3500 Hasselt, Belgium; Imec,\ncation-\u03c0 interaction. The metabolic stability of a short peptide, Imo-imomec, Hasselt University, B-3590 Diepenbeek,\nas well as its specificity in binding to the \u03b2-tubulin component Belgium; orcid.org/0000-0002-2264-2536;\nof a matured microtubule (MT) constitute the rationale for Email: anitha.ethirajan@uhasselt.be\nopting for the NAP peptide. Specific binding to the \u03b2-tubulin Batakrishna Jana \u2212 Department of Chemical Sciences and\nof an \u03b1, \u03b2-tubulin dimer is also examined using docking Center for Advanced Functional Materials, Indian Institute of\n Science Education and Research (IISER) Kolkata, Mohanpur\nstudies. This confirms stronger interactions between the Ru-\n 741246 West Bengal, India; Email: bkjdcs88@iiserkol.ac.in\nNAP peptide and \u03b2-tubulin compared to NAP. Efficient\n Benu Brata Das \u2212 Laboratory of Molecular Biology, School of\nbinding with Ru-NAP is also established using appropriate\n Biological Sciences, Indian Association for the Cultivation of\nbinding studies (KRu\u2011NAP: (6.8 \u00b1 0.55) \u00d7 106 M\u22121 and KNAP:\n Science, Jadavpur, Kolkata 700032 West Bengal, India;\n(8.2 \u00b1 1.1) \u00d7 104 M\u22121). The average linear charge density of\n orcid.org/0000-0003-2519-7105; Email: pcbbd@\n\u223c85 e/nm of MT further favors interaction with the cationic\n iacs.res.in\nRubpy part of a Ru-NAP conjugate accounts for the higher\n Amitava Das \u2212 Department of Chemical Sciences and Center\nbinding constant. Studies with anti-tubulin primary (EP1332Y\n for Advanced Functional Materials, Indian Institute of Science\n(1:300; v/v)) and secondary antibody (ab175471, 1:500 (v/\n Education and Research (IISER) Kolkata, Mohanpur\nv)) with MDA-MB-231 cells, pretreated with Ru-NAP and\n 741246 West Bengal, India; orcid.org/0000-0003-3666-\nfollowed by irradiation for 5 min (450 nm, 200 mW\u00b7cm\u22122, 5\n 1743; Email: amitava@iiserkol.ac.in\nmin, 60 J\u00b7cm\u22122) and then incubation in the dark for 20 h,\nrevealed a noticeable change in the cell cycle profile and cell Authors\ndeath. Tubulin polymerization assay (BK006P) confirmed that Atin Chatterjee \u2212 Department of Chemical Sciences and\nRu-NAP is effective in stabilizing MT and tubulin polymer- Center for Advanced Functional Materials, Indian Institute of\nization. These account for cell death primarily through cycle Science Education and Research (IISER) Kolkata, Mohanpur\narrest in the G2/M phase and cell apoptosis. Apart from the in 741246 West Bengal, India; Institute for Materials Research\nsitu generation of organelle-specific generation of cytotoxic (Imo-imomec), Nanobiophysics and Soft Matter Interfaces\n1\n O2, fiber formation on the MT surface and/or inside the (NSI) Group, Hasselt University, B-3500 Hasselt, Belgium\ntubular structure of MT would induce an MT dysfunction and Sandip Sarkar \u2212 Department of Chemical Sciences and Center\ncell death. Control studies reveal that the much lower toxicity for Advanced Functional Materials, Indian Institute of Science\nof Ru-NAP toward HEK293 cells compared to MDA-MB-231 Education and Research (IISER) Kolkata, Mohanpur\ncells (LightIC50Ru\u2011NAP[HEK293]: 17.2 \u00b1 2.5 \u03bcM, compared to 741246 West Bengal, India\nLight\n IC 50 Ru\u2011NAP [MDA-MB-231]: 32.5 \u00b1 7.8 nM, Dark I- Sangheeta Bhattacharjee \u2212 Laboratory of Molecular Biology,\n Ru\u2011NAP\nC 50 [HEK293]: > 80 \u03bcM compared to Dark I- School of Biological Sciences, Indian Association for the\n Ru\u2011NAP\nC50 [MDA-MB-231]: 2.9 \u00b1 0.5 \u03bcM) lies in the Cultivation of Science, Jadavpur, Kolkata 700032 West\naccelerated metabolic rate of MDA-MB-231 cells as a result Bengal, India\nof the Warburg effect coupled with the accumulation of small Arpan Bhattacharyya \u2212 Laboratory of Molecular Biology,\nmolecules in cells once they have formed aggregates. School of Biological Sciences, Indian Association for the\nImportantly, the Hill plot suggests a cooperative influence of Cultivation of Science, Jadavpur, Kolkata 700032 West\nfiber formation on binding to \u03b2-tubulin and 1O2 generation in Bengal, India\n 543 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n Surajit Barman \u2212 Department of Chemical Sciences and A. R.; Distel, M.; Thorn-Seshold, J.; Akhmanova, A.; Thorn-Seshold,\n Center for Advanced Functional Materials, Indian Institute of O. In Vivo Photocontrol of Microtubule Dynamics and Integrity,\n Science Education and Research (IISER) Kolkata, Mohanpur Migration and Mitosis, by The Potent GFP-Imaging-Compatible\n 741246 West Bengal, India Photoswitchable Reagents SBTubA4P and SBTub2M. J. Am. Chem.\n Soc. 2022, 144, 5614\u22125628.\n Uttam Pal \u2212 Technical Research Centre, S. N. Bose National\n (10) Klute, K.; Nackos, E.; Tasaki, S.; Nguyen, D. P.; Bander, N. H.;\n Centre for Basic Sciences, Salt Lake, Kolkata 700106, India; Tagawa, S. T. Microtubule Inhibitor-based Antibody-drug Conjugates\n orcid.org/0000-0003-2110-4610 for Cancer Therapy. Onco Targets Ther. 2014, 7, 2227\u22122236.\n Raviranjan Pandey \u2212 Department of Biological Sciences, (11) Mukhtar, E.; Adhami, V. M.; Mukhtar, H. Targeting\n Indian Institute of Science Education and Research (IISER) Microtubules by Natural Agents for Cancer Therapy. Mol. Cancer\n Kolkata, Mohanpur 741246 West Bengal, India Ther. 2014, 13, 275\u2212284.\nComplete contact information is available at: (12) Jordan, M. A.; Wilson, L. Microtubules as a Target for\n Anticancer Drugs. Nat. Rev. Cancer. 2004, 4, 253\u2212265.\nhttps://pubs.acs.org/10.1021/jacs.4c11820\n (13) P\u00e9rez-P\u00e9rez, M. J.; Priego, E. M.; Bueno, O.; Martins, M. S.;\n Canela, M. D.; Liekens, S. Blocking Blood Flow to Solid Tumors by\nAuthor Contributions Destabilizing Tubulin: an Approach to Targeting Tumor Growth. J.\n\u2207\n S.B. and A.B. are contributed equally. Med. Chem. 2016, 59, 8685\u22128711.\nNotes (14) Greene, L. M.; Meegan, M. J.; Zisterer, D. M. Combretastatins:\nThe authors declare no competing financial interest. More than just Vascular Targeting Agents? J. Pharmacol. Exp. Ther.\n 2015, 355, 212\u2212227.\n\u25a0 ACKNOWLEDGMENTS\nA.C. thanks the CSIR for a fellowship and the BOF-BILA\n (15) Xiao, H.; Verdier-Pinard, P.; Fernandez-Fuentes, N.; Burd, B.;\n Angeletti, R.; Fiser, A.; Horwitz, S. B.; Orr, G. A. Insights into the\n Mechanism of Microtubule Stabilization by Taxol. Proc. Natl. Acad.\nprogram (BOF21BL04) of Hasselt University. A.C. acknowl- Sci. U. S. A. 2006, 103, 10166\u221210173.\nedges IISER Kolkata and Nanobiophysics and Soft Matter (16) Markman, M. Antineoplastic Agents in the Management of\nInterfaces Laboratory, IMO-IMOMEC, Hasselt University for Ovarian Cancer: Current Status and Emerging Therapeutic Strategies.\nthe infrastructure. A.D. acknowledges the SERB-J.C. Bose Trends Pharmacol. Sci. 2008, 29, 515\u2212519.\nFellowship and funding through the JBR/2023/000005 grant. (17) Sharifi-Rad, J.; Quispe, C.; Patra, J. K.; Singh, Y. D.; Panda, M.\nA.D. also acknowledges the MoE-STARS research grant (No. K.; Das, G.; Adetunji, C. O.; Michael, O. S.; Sytar, O.; Polito, L.;\n2023-47) for financial support. B.J. acknowledges the SERB- Z\u030c ivkovic\u0301, J.; Cruz-Martins, N.; Klimek-Szczykutowicz, M.; Ekiert, H.;\nRamanujan Fellowship (RJF/2022/000127) for support. Choudhary, M. I.; Ayatollahi, S. A.; Tynybekov, B.; Kobarfard, F.;\n Muntean, A. C.; Grozea, I.; Das\u0327tan, S. D.; Butnariu, M.; Szopa, A.;\nB.B.D. team is supported by the SERB core research grant\n Calina, D. Paclitaxel: Application in Modern Oncology and\n(CRG/2022/001322), BRNS grant (54/14/10/2022-BRNS/ Nanomedicine-Based Cancer Therapy. Oxid. Med. Cell. Longev.\n11014), and ICMR grant (2021-11299/CMB/ADHOC- 2021, No. 3687700.\nBMS). S.B. acknowledges ICMR-RA Fellowship (45/35/ (18) Rohena, C. C.; Mooberry, S. L. Recent Progress with\n2022-DDI/BMS). Microtubule Stabilizers: New Compounds, Binding Modes and\n\n\u25a0 REFERENCES\n (1) Risinger, A. L.; Giles, F. J.; Mooberry, S. L. Microtubule\n Cellular Activities. Nat. Prod. Rep. 2014, 31, 335\u2212355.\n (19) Rixel, V. H. S. V.; Ramu, V.; Auyeung, A. B.; Beztsinna, N.;\n Leger, D. Y.; Lameijer, L. N.; Hilt, S. T.; D\u00e9v\u00e9dec, S. E. L.; Yildiz, T.;\nDynamics as a Target in Oncology. Cancer Treat. Rev. 2009, 35, 255\u2212 Betancourt, T.; Gildner, M. B.; Hudnall, T. W.; Sol, V.; Liagre, B.;\n261. Kornienko, A.; Bonnet, S. Photo-Uncaging of a Microtubule-Targeted\n (2) Weaver, B. A. How Taxol/Paclitaxel Kills Cancer Cells. Mol. Biol. Rigidin Analogue in Hypoxic Cancer Cells and in a Xenograft Mouse\nCell 2014, 25, 2677\u22122681. Model. J. Am. Chem. Soc. 2019, 141, 18444\u221218454.\n (3) Wang, X.; Gigant, B.; Zheng, X.; Chen, Q. Microtubule-targeting (20) Divinski, I.; Holtser-Cochav, M.; Vulih-Schultzman, I.;\nAgents for Cancer Treatment: Seven Binding Sites and Three Steingart, R. A.; Gozes, I. Peptide Neuroprotection through Specific\nStrategies. MedComm Oncol. 2023, 2, No. e46. Interaction with Brain Tubulin. J. Neurochem. 2006, 98, 973\u2212984.\n (4) Zheng, L.; Ren, R.; Sun, X.; Zou, Y.; Shi, Y.; Di, B.; Niu, M.-M. (21) Oz, S.; Kapitansky, O.; Ivashco-Pachima, Y.; Malishkevich, A.;\nDiscovery of a Dual Tubulin and Poly (ADP-Ribose) Polymerase-1 Giladi, E.; Skalka, N.; Rosin-Arbesfeld, R.; Mittelman, L.; Segev, O.;\nInhibitor by Structure-Based Pharmacophore Modeling, Virtual Hirsch, J. A.; Gozes, I. The NAP Motif of Activity-Dependent\nScreening, Molecular Docking, and Biological Evaluation. J. Med. Neuroprotective Protein (ADNP) Regulates Dendritic Spines through\nChem. 2021, 64 (21), 15702\u221215715. Microtubule End Binding Proteins. Mol. Psychiatry. 2014, 19, 1115\u2212\n (5) Shuai, W.; Wang, G.; Zhang, Y.; Bu, F.; Zhang, S.; Miller, D. D.; 1124.\nLi, W.; Ouyang, L.; Wang, Y. Recent Progress on Tubulin Inhibitors (22) Li, Y.; Zhang, H.; Merkher, Y. Recent Advances in Therapeutic\nwith Dual Targeting Capabilities for Cancer Therapy. J. Med. Chem. Strategies for Triple-Negative Breast Cancer. J. Hematol. Oncol. 2022,\n2021, 64 (12), 7963\u22127990. 15, 121.\n (6) Dumontet, C.; Jordan, M. A. Microtubule-binding Agents: A (23) Bulina, M. E.; Lukyanov, K. A.; Britanova, O. V.; Onichtchouk,\nDynamic Field of Cancer Therapeutics. Nat. Rev. Drug Discovery D.; Lukyanov, S.; Chudakov, D. M. Chromophore-Assisted Light\n2010, 9, 790\u2212803. Inactivation (CALI) using the Phototoxic Fluorescent Protein\n (7) Panda, D.; Rathinasamy, K.; Santra, M. K.; Wilson, L. Kinetic KillerRed. Nat. Protoc. 2006, 1, 947\u2212953.\nSuppression of Microtubule Dynamic Instability by Griseofulvin: (24) Zhang, L.; Wang, P.; Zhou, X.-Q.; Bretin, L.; Zeng, X.; Husiev,\nImplications for its Possible Use in the Treatment of Cancer. Proc. Y.; Polanco, E. A.; Zhao, G.; Wijaya, L. S.; Biver, T.; Le D\u00e9v\u00e9dec, S.\nNatl. Acad. Sci. U. S. A. 2005, 102, 9878\u22129883. E.; Sun, W.; Bonnet, S. Cyclic Ruthenium-Peptide Conjugates as\n (8) Honore, S.; Pasquier, E.; Braguer, D. Understanding Micro- Integrin-Targeting Phototherapeutic Prodrugs for the Treatment of\ntubule Dynamics for Improved Cancer Therapy. Cell. Mol. Life Sci. Brain Tumors. J. Am. Chem. Soc. 2023, 145, 14963\u221214980.\n2005, 62, 3039\u22123056. (25) Cole, H. D.; Vali, A.; Roque, J. A.; Shi, G.; Kaur, G.; Hodges, R.\n (9) Gao, L.; Meiring, J. C. M.; Varady, A.; Ruider, I. E.; Heise, C.; O.; Franc\u00e9s-Monerris, A.; Alberto, M. E.; Cameron, C. G.; McFarland,\nWranik, M.; Velasco, C. D.; Taylor, J. A.; Terni, B.; Weinert, T.; S. A. Ru (II) Phenanthroline-Based Oligothienyl Complexes as\nStandfuss, J.; Cabernard, C. C.; Llobet, A.; Steinmetz, M. O.; Bausch, Phototherapy Agents. Inorg. Chem. 2024, 63, 11450\u221211458.\n\n 544 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n (26) Burke, C. S.; Byrne, A.; Keyes, T. E. Targeting Photoinduced (45) Sailer, A.; Meiring, J. C. M.; Heise, C.; Pettersson, L. N.;\nDNA Destruction by Ru(II) Tetraazaphenanthrene in Live Cells by Akhmanova, A.; Thorn-Seshold, J.; Thorn-Seshold, O. Pyrrole\nSignal Peptide. J. Am. Chem. Soc. 2018, 140, 6945\u22126955. Hemithioindigo Antimitotics with Near-Quantitative Bidirectional\n (27) Chakrabortty, S.; Agrawalla, B. K.; Stumper, A.; Vegi, N. M.; Photoswitching that Photocontrol Cellular Microtubule Dynamics\nFischer, S.; Reichardt, C.; K\u00f6gler, M.; Dietzek, B.; Feuring-Buske, M.; with Single-Cell Precision. Angew. Chem., Int. Ed. 2021, 60, 23695.\nBuske, C.; Rau, S.; Weil, T. Mitochondria Targeted Protein- (46) Elgar, C. E. E.; Yusoh, N. A.; Tiley, P. R.; Kolozsv\u00e1ri, N.;\nRuthenium Photosensitizer for Efficient Photodynamic Applications. Bennett, L. G.; Gamble, A.; P\u00e9an, E. V.; Davies, M. L.; Staples, C. J.;\nJ. Am. Chem. Soc. 2017, 139, 2512\u22122519. Ahmad, H.; Gill, M. R. Ruthenium (II) Polypyridyl Complexes as\n (28) Jacobson, K.; Rajfur, Z.; Vitriol, E.; Hahn, K. Chromophore- FRET Donors: Structure-and Sequence-Selective DNA-Binding and\nAssisted Laser Inactivation in Cell Biology. Trends Cell Biol. 2008, 18, Anticancer Properties. J. Am. Chem. Soc. 2023, 145, 1236\u22121246.\n443\u2212450. (47) B, J.; Zhang, Y.; Wu, S.; Li, H.; Sun, L.; Liu, Y.; Zhu, X.; Qiao,\n (29) Ogorek, A. N.; Zhou, X.; Martell, J. D. Switchable DNA X.; Ma, Q.; Liu, C.; Niu, N.; Xue, J.; Chen, G.; Yang, Y.; Liu, C. KK-\nCatalysts for Proximity Labeling at Sites of Protein-Protein LC-1 as a Therapeutic Target to Eliminate ALDH+ Stem Cells in\nInteractions. J. Am. Chem. Soc. 2023, 145, 16913\u221216923. Triple Negative Breast Cancer. Nat. Commun. 2023, 14, 2602.\n (30) Sato, S.; Morita, K.; Nakamura, H. Regulation of Target Protein (48) Maisch, T.; Baier, J.; Franz, B.; B\u00e4umler, W. The Role of Singlet\nKnockdown and Labeling Using Ligand-Directed Ru(bpy)3 Photo- Oxygen and Oxygen Concentration in Photodynamic Inactivation of\ncatalyst. Bioconjugate Chem. 2015, 26, 250\u2212256. Bacteria. Proc. Natl. Acad. Sci. U. S. A. 2007, 104, 7223\u22127228.\n (31) Takemoto, K.; Iwanari, H.; Tada, H.; Suyama, K.; Sano, A.; (49) Carbonaro, M.; Escuin, D.; O\u2019Brate, A.; Thadani-Mulero, M.;\nNagai, T.; Hamakubo, T.; Takahashi, T. Optical Inactivation of Giannakakou, P. Microtubules Regulate Hypoxia-inducible Factor-1\u03b1\nSynaptic AMPA Receptors Erases Fear Memory. Nat. Biotechnol. Protein Trafficking and Activity. J. Biol. Chem. 2012, 287, 11859\u2212\n2017, 35, 38\u221247. 11869.\n (32) Robert, A.; Hill, R. A.; Eyiyemisi, C.; Damisah, E. C.; Chen, F.; (50) Raza, A.; Archer, S. A.; Fairbanks, S. D.; Smitten, K. L.;\nAlex, C.; Kwan, A. C.; Grutzendler, J. Targeted Two-Photon Botchway, S. W.; Thomas, J. A.; MacNeil, S.; Haycock, J. W. A\nChemical Apoptotic Ablation of Defined Cell Types in Vivo. Nat. Dinuclear Ruthenium (II) Complex Excited by Near-Infrared Light\nCommun. 2017, 8, No. 15837. through Two-Photon Absorption Induces Phototoxicity Deep within\n (33) Fu, Z.; Li, S.; Han, S. Antibody Drug Conjugate: the \u201cBiological Hypoxic Regions of Melanoma Cancer Spheroids. J. Am. Chem. Soc.\nMissile\u201d for Targeted Cancer Therapy. Signal Transduction Targeted 2020, 142, 4639\u22124647.\nTher. 2022, 7, 93. (51) Heinemann, F.; Karges, J.; Gasser, G. Critical Overview of the\n (34) Beck, A.; Goetsch, L.; Dumontet, C.; Corva\u00efa, N. Strategies and Use of Ru(II) Polypyridyl Complexes as Photosensitizers in One-\nChallenges for the Next Generation of Antibody-Drug Conjugates. Photon and Two-Photon Photodynamic Therapy. Acc. Chem. Res.\nNat. Rev. Drug Discovery 2017, 16, 315\u2212337. 2017, 50, 2727\u22122736.\n (35) Acharya, S.; Maji, M.; Chakraborty, M. P.; Bhattacharya, I.; (52) Li, X.; Kwon, N.; Guo, T.; Liu, Z.; Yoon, J. Innovative\n Strategies for Hypoxic-Tumor Photodynamic Therapy. Angew. Chem.,\nDas, R.; Gupta, A.; Mukherjee, A. Disruption of the Microtubule\n Int. Ed. 2018, 57, 11522\u221211531.\nNetwork and Inhibition of VEGFR2 Phosphorylation by Cytotoxic\n (53) Ghosh, A.; Das, P.; Gill, M. R.; Kar, P.; Walker, M. G.; Thomas,\nN,O-Coordinated Pt(II) and Ru(II) Complexes of Trimethoxy\n J. A.; Das, A. Photoactive RuII-Polypyridyl Complexes that Display\nAniline-Based Schiff Bases. Inorg. Chem. 2021, 60, 3418\u22123430.\n Sequence Selectivity and High-Affinity Binding to Duplex DNA\n (36) Adak, A.; Mohapatra, S.; Mondal, P.; Jana, B.; Ghosh, S. Design\n through Groove Binding. Chem.\ufffdEur. J. 2011, 17, 2089\u22122098.\nof a Novel Microtubule Targeted Peptide Vesicle for Delivering\n (54) Poynton, F. E.; Bright, S. A.; Blasco, S.; Williams, D. C.; Kelly,\nDifferent Anticancer Drugs. Chem. Commun. 2016, 52, 7549\u22127552. J. M.; Gunnlaugsson, T. The Development of Ruthenium (II)\n (37) Gong, L.; Zhao, H.; Liu, Y.; Wu, H.; Liu, C.; Chang, S.; Chen, Polypyridyl Complexes and Conjugates for in Vitro Cellular and in\nL.; Jin, M.; Wang, Q.; Gao, Z.; Huang, W. Research Advances in Vivo Applications. Chem. Soc. Rev. 2017, 46, 7706\u22127756.\nPeptide-Drug Conjugates. Acta Pharm. Sin. B 2023, 13, 3659\u22123677. (55) Bonnet, S. Ruthenium-Based Photoactivated Chemotherapy. J.\n (38) Rizvi, S. F. A.; Zhang, L.; Zhang, H.; Fang, Q. Peptide-Drug Am. Chem. Soc. 2023, 145, 23397\u221223415.\nConjugates: Design, Chemistry, and Drug Delivery System as a Novel (56) Goodson, H. V.; Jonasson, E. M. Microtubules and Micro-\nCancer Theranostic. ACS Pharmacol. Transl. Sci. 2024, 7, 309\u2212334. tubule-Associated Proteins. Cold Spring Harb. Perspect. Biol. 2018, 10,\n (39) Plaetzer, K.; Krammer, B.; Berlanda, J.; Berr, F.; Kiesslich, T. a022608.\nPhotophysics and photochemistry of photodynamic therapy: (57) Sekulic\u0301, D. L.; Sataric\u0301, M. V. An Improved Nanoscale\nfundamental aspects. Lasers. Med. Sci. 2009, 24, 259\u2212268. Transmission Line Model of Microtubule: The Effect of Nonlinearity\n (40) Ghosh, H. N.; Das, A. Advances in Inorganic Chemistry; on the Propagation of Electrical Signals. Elec. Energy 2015, 28, 133\u2212\nAcademic Press-ELSEVIER, 2023; Vol. 81, pp 305\u2212344. 142.\n (41) Yi, X.; Dai, J.; Han, Y.; Xu, M.; Zhang, X.; Zhen, S.; Zhao, Z.; (58) Minoura, I.; Muto, E. Dielectric Measurement of Individual\nLou, X.; Xia, F. A high therapeutic efficacy of polymeric prodrug Microtubules Using the Electroorientation Method. Biophys. J. 2006,\nnano-assembly for a combination of photodynamic therapy and 90, 3739\u22123748.\nchemotherapy. Commun. Biol. 2018, 1, 202. (59) Adak, A.; Das, G.; Barman, S.; Mohapatra, S.; Bhunia, D.; Jana,\n (42) Bretin, L.; Husiev, Y.; Ramu, V.; Zhang, L.; Hakkennes, M.; B.; Ghosh, S. Biodegradable Neuro-Compatible Peptide Hydrogel\nAbyar, S.; Johns, A. C.; D\u00e9v\u00e9dec, S. E. L.; Betancourt, T.; Kornienko, Promotes Neurite Outgrowth, Shows Significant Neuroprotection,\nA.; Bonnet, S. Red-Light Activation of a Microtubule Polymerization and Delivers Anti-Alzheimer Drug. ACS Appl. Mater. Interfaces. 2017,\nInhibitor via Amide Functionalization of the Ruthenium Photocage. 9, 5067\u22125076.\nAngew. Chem., Int. Ed. 2024, 63, No. e202316425. (60) Pierce, S.; Jennings, M. P.; Juliano, S. A.; Angeles-Boza, A. M.\n (43) Cao, F.; Wang, H.; Lu, N.; Zhang, P.; Huang, H. A Peptide-Ruthenium Conjugate as an Efficient Photosensitizer for the\nPhotoisomerizable Zinc (II) Complex Inhibits Microtubule Polymer- Inactivation of Multidrug-Resistant Bacteria. Inorg. Chem. 2020, 59,\nization for Photoactive Therapy. Angew. Chem., Int. Ed. 2023, 62, 14866\u221214870.\nNo. e202301344. (61) Kandoth, N.; Chaudhary, S.; Gupta, S.; Raksha, K.; Chatterjee,\n (44) Schmitt, C.; Mauker, P.; Vepr\u030cek, N. A.; Gierse, C.; Meiring, J. A.; Gupta, S.; Karuthedath, S.; Castro, C. D.; Laquai, F.; Pramanik, S.\nC. M.; Kuch, J.; Akhmanova, A.; Dehmelt, L.; Thorn-Seshold, O. A K.; Bhattacharyya, S.; Mallick, A. I.; Das, A. Multimodal Biofilm\nPhotocaged Microtubule-Stabilising Epothilone Allows Spatiotempo- Inactivation Using a Photocatalytic Bismuth Perovskite-TiO2-Ru(II)-\nral Control of Cytoskeletal Dynamics. Angew. Chem., Int. Ed. 2024, 63, polypyridyl-Based Multisite Heterojunction. ACS Nano 2023, 17,\nNo. e202410169. 10393\u221210406.\n\n 545 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\n (62) Banerjee, T.; Rawalekar, S.; Das, A.; Ghosh, H. N. Interfacial (79) Han, G.; Li, G.; Huang, J.; Han, C.; Turro, C.; Sun, Y. Two-\nElectron Transfer Dynamics of Two Newly Synthesized Catecholate Photon-Absorbing Ruthenium Complexes Enable Near Infrared\nBound RuII Polypyridyl-Based Sensitizers on TiO2 Nanoparticle Light-Driven Photocatalysis. Nat. Commun. 2022, 13, 2288.\nSurface-A Femtosecond Pump Probe Spectroscopic Study. Eur. J. (80) Toupin, N.; Steinke, S. J.; Nadella, S.; Li, A.; Rohrabaugh, T.\nInorg. Chem. 2011, 2011, 4187\u22124197. N.; Samuels, E. R.; Turro, C.; Sevrioukova, I. F.; Kodanko, J. J.\n (63) Mahato, P.; Saha, S.; Choudhury, S.; Das, A. Solvent- Photosensitive Ru(II) Complexes as Inhibitors of the Major Human\nDependent Aggregation Behavior of a New Ru (II)-Polypyridyl Drug Metabolizing Enzyme CYP3A4. J. Am. Chem. Soc. 2021, 143,\nBased Metallo surfactant. Chem. Commun. 2011, 47, 11074\u221211076. 9191\u22129205.\n (64) Zhang, K. Y.; Yu, Q.; Wei, H.; Liu, S.; Zhao, Q.; Huang, W. (81) Acharya, S.; Maji, M.; Chakraborty, M. P.; Bhattacharya, I.;\nLong-Lived Emissive Probes for Time-Resolved Photoluminescence Das, R.; Gupta, A.; Mukherjee, A. Disruption of Microtubule Network\nBioimaging and Biosensing Chem. Rev. 2018, 118, 1770\u22121839. and Inhibition of VEGFR2 Phosphorylation by Cytotoxic N,O\n (65) Shum, J.; Leung, P. K-K.; Lo, K. K-W. Luminescent Ruthenium Coordinated Pt(II) and Ru(II) Complexes of Trimethoxy Aniline\n(II) Polypyridine Complexes for a Wide Variety of Biomolecular and Based Schiff Bases. Inorg. Chem. 2021, 60, 3418\u22123430.\nCellular Applications. Inorg. Chem. 2019, 58, 2231\u22122247. (82) van Rijt, S. H.; Mukherjee, A.; Pizarro, A. M.; Sadler, P. J.\n (66) Byrne, A.; Burkeab, C. S.; Keyes, T. E. Precision Targeted Cytotoxicity, Hydrophobicity, Uptake, and Distribution of Osmium-\nRuthenium (II) Luminophores; Highly Effective Probes for Cell (II) Anticancer Complexes in Ovarian Cancer Cells. J. Med. Chem.\nImaging by Stimulated Emission Depletion (STED) Microscopy. 2010, 53, 840\u2212849.\nChem. Sci. 2016, 7, 6551\u22126562. (83) Zhang, R.; Qin, X.; Kong, F.; Chen, P.; Pan, G. Improving\n (67) Martin, A.; Byrne, A.; Burke, C. S.; Forster, R. J.; Keyes, T. E. Cellular Uptake of Therapeutic Entities through Interaction with\nPeptide-Bridged Dinuclear Ru (II) Complex for Mitochondrial Components of Cell Membrane. Drug Delivery 2019, 26, 328\u2212342.\nTargeted Monitoring of Dynamic Changes to Oxygen Concentration (84) Mitchell, M. J.; Billingsley, M. M.; Haley; Wechsler, M. E.;\nand ROS Generation in Live Mammalian Cells. J. Am. Chem. Soc. Peppas, N. A.; Langer, R. Engineering Precision Nanoparticles for\n2014, 136, 15300\u221215309. Drug Delivery. Nat. Rev. Drug Discovery 2021, 20, 101\u2212124.\n (68) Gozes, I.; Morimoto, B. H.; Tiong, J.; Fox, A.; Sutherland, K.; (85) Bhargava, S.; Kulkarni, R.; Dewangan, B.; Jiaswar, C.; Kumar,\nDangoor, D.; Holser-Cochav, M.; Vered, K.; Newton, P.; Aisen, P. S.; K.; Kumar, A.; Kulkarni, N.; Bodhe, P. R.; Kumarb, H.; Sahu, B.\nMatsuoka, Y.; Dyck, C. H. V.; Thal, L. NAP: Research and Microtubule Stabilising Peptides: New Paradigm towards Manage-\nDevelopment of a Peptide Derived from Activity-Dependent ment of Neuronal Disorders. RSC Med. Chem. 2023, 14, 2192\u22122205.\nNeuroprotective Protein (ADNP). CNS Drug Rev. 2005, 11, 353\u2212 (86) Silva, S.; Almeida, A. J.; Vale, N. Combination of Cell-\n Penetrating Peptides with Nanoparticles for Therapeutic Application:\n368.\n (69) Yammine, A.; Gao, J.; Kwan, A. H. Tryptophan Fluorescence A Review. Biomol. 2019, 9, 22.\n (87) Liu, K.; Newbury, P. A.; Glicksberg, B. S.; Zeng, W. Z. D.;\nQuenching Assays for Measuring Protein-ligand Binding Affinities:\n Paithankar, S.; Andrechek, E. R.; Chen, B. Evaluating Cell Lines as\nPrinciples and a Practical Guide. Bio Protoc. 2019, 9, No. e3253.\n Models for Metastatic Breast Cancer through Integrative Analysis of\n (70) Chakraborti, S.; Das, L.; Kapoor, A. N.; Dwivedi, V.; Poddar,\n Genomic Data. Nat. Commun. 2019, 10, 2138.\nA.; Chakraborti, G.; Janik, M.; Basu, G.; Panda, D.; Chakrabarti, P.;\n (88) Gao, Y.; Shi, J.; Yuan, D.; Xu, B. Imaging Enzyme-Triggered\nSurolia, A.; Bhattacharyya, B. Curcumin Recognizes a Unique Binding\n Self-Assembly of Small Molecules inside Live Cells. Nat. Commun.\nSite of Tubulin. J. Med. Chem. 2011, 54, 6183\u22126196.\n 2012, 3, 1033.\n (71) Jana, B.; Mohapatra, S.; Mondal, P.; Barman, S.; Pradhan, K.;\n (89) Bigman, L. S.; Levy, Y. Protein Diffusion on Charged\nSaha, A.; Ghosh, A. \u03b1-Cyclodextrin Interacts Close to Vinblastine Site\n Biopolymers: DNA Versus Microtubule. Biophys. J. 2020, 118,\nof Tubulin and Delivers Curcumin Preferentially to the Tubulin 3008\u22123018.\nSurface of Cancer Cell. ACS Appl. Mater. Interfaces. 2016, 8, 13793\u2212 (90) Hsu, P. P.; Sabatini, D. M. Cancer Cell Metabolism: Warburg\n13803. and Beyond. Cell. 2008, 134, 703\u2212707.\n (72) Gupta, K.; Panda, D. Perturbation of Microtubule Polymer- (91) Yang, Z. M.; Liang, G. L.; Guo, Z. F.; Guo, Z. H.; Xu, B.\nization by Quercetin through Tubulin Binding: A Novel Mechanism Intracellular Hydrogelation of Small Molecules Inhibits Bacterial\nof its Antiproliferative Activity. Biochemistry. 2002, 41, 13029\u221213038. Growth. Angew. Chem., Int. Ed. 2007, 46, 8216\u22128219.\n (73) Biswas, A.; Kurkute, P.; Jana, B.; Laskar, A.; Ghosh, S. An (92) Kuang, Y.; Xu, B. Disruption of the Dynamics of Microtubules\nAmyloid Inhibitor Octapeptide forms Amyloid Type Fibrous and Selective Inhibition of Glioblastoma Cells by Nanofibers of Small\nAggregates and Affects Microtubule Motility. Chem. Commun. 2014, Hydrophobic Molecules. Angew. Chem., Int. Ed. 2013, 52, 6944\u22126948.\n50, 2604\u22122607. (93) D\u2019Amato, A.; Mariconda, A.; Iacopetta, D.; Ceramella, J.;\n (74) Ashur-Fabian, O.; Segal-Ruder, Y.; Skutelsky, E.; Brenneman, Catalano, A.; Sinicropi, M. S.; Longo, P. Complexes of Ruthenium(II)\nD. E.; Steingart, R. A.; Giladi, E.; Gozes, I. The Neuroprotective as Promising Dual-Active Agents against Cancer and Viral Infections.\nPeptide NAP Inhibits the Aggregation of the Beta-Amyloid Peptide. Pharmaceuticals. 2023, 16, 1729.\nPeptides 2003, 24, 1413\u22121423. (94) Verlato, G.; Cerveri, I.; Villani, A.; Pasquetto, M.; Ferrari, M.;\n (75) Przybyla, D. E.; Chmielewski, J. Metal-Triggered Radial Self- Fanfulla, F.; Zanolin, E.; Rijcken, B.; Marco, R. Evaluation of\nAssembly of Collagen Peptide fibers. J. Am. Chem. Soc. 2008, 130, Methacholine Dose-Response Curves by Linear and Exponential\n12610\u221212611. Mathematical Models: Goodness-of-Fit and Validity of Extrapolation.\n (76) Entradas, T.; Waldrona, S.; Volk, M. The Detection Sensitivity Eur. Respir. J. 1996, 9, 506\u2212511.\nof Commonly Used Singlet Oxygen Probes in Aqueous Environ- (95) Wang, C.; Huang, S.-B.; Yang, M.-C.; Lin, Y.-T.; Chu, I.-H.;\nments. J. Photochem. Photobiol., B 2020, 204, No. 111787. Shen, Y.-N.; Chiu, Y.-H.; Hung, S.-H.; Kang, L.; Hong, Y.-R.\n (77) Lindig, B. A.; Rodgers, M. A. J.; Paul Schaap, A. Determination Combining Paclitaxel with ABT-263 Has a Synergistic Effect on\nof the Lifetime of Singlet Oxygen in D2O Using 9,10-Anthracenedi- Paclitaxel Resistant Prostate Cancer Cells. PLoS One. 2015, 10,\npropionic Acid, a Water-Soluble Probe. J. Am. Chem. Soc. 1980, 102, No. e0120913.\n5590\u22125593. (96) De, K. Decapeptide Modified Doxorubicin Loaded Solid Lipid\n (78) Ramu, V.; Aute, S.; Taye, N.; Guha, R.; Walker, M. G.; Mogare, Nanoparticles as Targeted Drug Delivery System against Prostate\nD.; Parulekar, A.; Thomas, J. A.; Chattopadhyay, S.; Das, A. Photo- Cancer. Langmuir. 2021, 37, 13194\u221213207.\nInduced Cytotoxicity and Anti-Metastatic Activity of Ruthenium(II)- (97) Sarkar, S.; Chatterjee, A.; Kim, D.; Saritha, C.; Barman, S.; Jana,\nPolypyridyl Complexes Functionalized with Tyrosine or Tryptophan. B.; Ryu, J.-H.; Das, A. Host-Guest Adduct as a Stimuli-responsive\nDalton Trans. 2017, 46, 6634\u22126644. Prodrug: Enzyme-Triggered Self-Assembly Process of a Short Peptide\n\n 546 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\fJournal of the American Chemical Society pubs.acs.org/JACS Article\n\nwithin Mitochondria to Induce Cell Apoptosis. Adv. Healthcare Mater.\n2024, No. 2403243.\n\n\n\n\n 547 https://doi.org/10.1021/jacs.4c11820\n J. Am. Chem. Soc. 2025, 147, 532\u2212547\n\f", "pages_extracted": 16, "text_length": 116201}