A radiation-responsive ruthenium (III)-based coordination complex and a method of preparation thereof

Abstract

The present invention discloses a radiation-responsive ruthenium (III)-based coordination complex and a method of preparation thereof. The pyrene-terpyridine-polyethylene glycol-functionalized ruthenium(III) trichloride complex, exhibits enhanced cytotoxicity toward cancer cells upon exposure to ionizing radiation, while maintains comparatively lower toxicity toward normal cells and demonstrates effective radiation-enhanced biological damage in radioresistant model systems.

Description

FIELD OF THE INVENTIONThe present invention relates to the field of coordination complex chemistry and cancerbiology. In particular, the present invention relates to a radiation-responsive ruthenium (III) -based coordination complex and its application as a selective radiosensitizing anticancer agent.More particularly, the present invention relates to a pyrene-terpyridine-polyethylene glycolfunctionalized ruthenium(III) trichloride complex, that exhibits enhanced cytotoxicity towardcancer cells upon exposure to ionizing radiation, while maintaining comparatively lowertoxicity toward normal cells and demonstrating effective radiation-enhanced biological damagein radioresistant model systems.BACKGROUND OF THE INVENTIONCancer is a complex group of diseases characterized by the uncontrolled growth, proliferation,and spread of abnormal cells within the body. These malignant cells evade normal regulatorymechanisms, acquire the ability to invade surrounding tissues, and may metastasize to distantorgans, ultimately impairing essential physiological functions. Despite decades of advancementin diagnostics and therapeutics, cancer remains one of the leading causes of morbidity andmortality worldwide. Its heterogeneity across tissue types, genetic mutations, and tumourmicroenvironments continues to pose significant challenges for effective, targeted, andaffordable treatment.Radiation therapy (RT) is a mainstay modality in the clinical management of many solidtumors. Although ionizing radiation can effectively kill tumor cells, the therapeutic index ofRT is often limited by (a) intrinsic or acquired tumor radioresistance, (b) collateral damage tosurrounding healthy tissues, and (c) dose-limiting toxicity which constrains escalation ofradiation dose. Strategies that increase tumor radiosensitivity without proportionally increasingnormal tissue toxicity are therefore highly desirable to improve local control and patientoutcomes.Several chemical radiosensitizers and metal coordination complexes have been investigated toenhance the biological effects of ionizing radiation. Among transition-metal based systems,ruthenium (II) polypyridyl complexes have emerged as promising candidates because offavorable redox chemistry, tunable photophysical properties, and the ability to targetsubcellular compartments (for example mitochondria or DNA). These complexes have beenstudied both as stand-alone chemotherapeutic agents and as photo / radiosensitizers incombination with light or ionizing radiation. Reviews and experimental studies illustrate thatstructural modification (choice of ligands, appended aromatic systems, and hydrophilic handlessuch as PEG) can markedly change cellular uptake, selectivity for cancer cells, and mechanismsof action (ROS generation, mitochondrial disruption, DNA damage).However, existing solutions show several limitations. First, many reported rutheniumcomplexes suffer from poor aqueous solubility or aggregation in biological media, whichreduces bioavailability and complicates formulation for systemic or local delivery. Second,several metal complexes that are phototoxic or redox-active in vitro display inadequateselectivity between cancer and normal cells; this narrow therapeutic window raises safetyconcerns for translational use. Third, while photodynamic therapy (PDT) rutheniumcompounds exploit light activation, light penetration is limited for deep tumors whereas X-rayactivation or radiation-triggered activation offers a route to treat deeper tissues but reports ofruthenium complexes explicitly designed and validated as X-ray radiosensitizers (withPEGylation and membrane-interacting aromatic groups) remain limited.Thus, there is an unmet need for water-soluble, radiation-responsive ruthenium complexes thatcombine selective radiosensitization with improved biocompatibility. Accordingly, there existsa critical need for radiation-activated therapeutic agents that can selectively enhance radiationinduced tumor cell killing at clinically relevant or reduced radiation doses.OBJECTIVES OF THE INVENTIONIn view of the foregoing disadvantages inherent in the existing arts, the primary objective ofthe present invention is to provide a ruthenium (III)-based coordination complex that functionsas a radiation-responsive radiosensitizing agent for enhancing the therapeutic efficacy ofionizing radiation in cancer treatment.Another objective of the present invention is to develop a radiosensitizer that exhibitspreferential cytotoxicity toward cancer cells under X-ray irradiation, while maintaining reducedtoxicity toward normal cells, thereby offering an improved therapeutic window duringradiotherapy.Yet another objective of the present invention is to provide a PEG-conjugated ruthenium (III)-based complex with improved aqueous solubility, biological stability, and biocompatibility,making it suitable for biological and therapeutic applications involving exposure to ionizingradiation.Another objective of the present invention is to validate the radiosensitizing capability ofruthenium (III)- based coordination complex thereby demonstrating its potential to overcomeextreme radiation tolerance mechanisms and supporting its application in radiation-assistedcancer therapy.Still another objective of the present invention is to provide a method for the synthesis ofruthenium (III)- based coordination complex.These and other objects and advantages of the present subject matter will be apparent to aperson skilled in the art after consideration of the following detailed description, taking intoconsideration accompanying drawings in which preferred embodiments of the present subjectmatter are illustrated.SUMMARY OF THE INVENTIONAn aspect of the present invention provides a Ruthenium (III)-based coordination complexhaving the structure formula IFormula Iwherein n - 9.Another aspect of the present invention provides a Ruthenium (III)-based coordination complextrichlorido{6,6''-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2''-terpyridine]-κ3N}ruthenium(III).Yet another aspect of the present invention provides a method for preparation of a Ruthenium(III) -based coordination complex having the structure formula IFormula Iwherein n - 9,comprising the steps:a. dissolving a ruthenium salt in a first solvent to obtain a solution;b. dissolving a ligand in a second solvent to obtain a ligand solution;c. adding the solution dropwise to the ligand solution to obtain a reaction mixture;d. refluxing the reaction mixture at a temperature in the range of 60-70°C for 8-10hours; ande. removing the solvents to obtain the Ruthenium (III) -based coordination complex.These and other aspects of the disclosed subject matter, as well as additional novel features,will be apparent from the description provided herein. The intent of this summary is not to bea comprehensive description of the claimed subject matter, but rather to provide a shortoverview of some of the subject matter's functionality. Other systems, methods, features andadvantages here provided will become apparent to one with skill in the art upon examinationof the following figures and detailed description. It is intended that all such additional systems,methods, features and advantages that are included within this description be within the scopeof any claims.BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS:It is to be noted, however, that the appended drawings illustrate only typical embodiments ofthe present subject matter and are therefore not to be considered for limiting its scope, for theinvention may admit to other equally effective embodiments. The detailed description isdescribed concerning the accompanying figures. Some embodiments of system or methods inaccordance with embodiments of the present subject matter are now described, by way ofexample,andwithreferencetotheaccompanyingfigures,inwhich:Figure 1 (a) illustrates UV-vis absorption spectra of Ruthenium complex, PTP-Ru, in solutionstate at 25°C, (b) Normalised Solution state fluorescence spectra of PTP-Ru at 25°C, (c) C.I.E.plot for PTP-Ru in solution state, and (d) FT-IR spectrum of PTP-Ru at 25°C (ATR method).Figure 2 illustrates the spot assay of Deinococcus radiodurans used as a radioresistant model,showing serial bacterial dilutions treated with PTP-Ru (1 mg / mL) in the absence and presenceof X-ray irradiation (50 Gy) to evaluate radiation-enhanced biological effects.Figure 3 illustrates the spread plate assay of Deinococcus radiodurans used as a radioresistantmodel, showing the effect of PTP-Ru (1 mg / mL) with and without X-ray irradiation (50 Gy)on colony formation.Figure 4 illustrates the cytotoxicity evaluation of PTP-Ru in HeLa cancer cells, comparing cellviability in the absence and presence of X-ray irradiation (15 Gy) to demonstrate radiationenhanced anticancer activity.Figure 5 illustrates the biocompatibility and cytotoxicity assessment of PTP-Ru in C2C12normal cells, showing cell viability in the absence and presence of X-ray irradiation (15 Gy) toevaluate selective radiosensitization.DETAILED DESCRIPTION OF THE INVENTIONA detailed description of various exemplary embodiments of the disclosure is described herein.It should be noted that the embodiments are described herein in such detail as to communicatethe disclosure. However, the amount of details provided herein is not intended to limit theanticipated variations of embodiments; on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spirit and scope of the presentdisclosure.It is also to be understood that various substitutions / arrangements / permutations orcombinations may be devised that, although not explicitly described or shown herein, embodythe principles of the present disclosure. Moreover, all statements herein reciting principles,aspects, and embodiments of the present disclosure, as well as specific examples, are intendedto encompass equivalents thereof.The terminology used herein is to describe particular embodiments only and is not intended tobe limiting to example embodiments. As used herein, the singular forms "a", "an" and "the"are intended to include the plural forms as well, unless the context indicates otherwise. It willbfhddhhiiiildd / ildiwhen used herein, specify the presence of stated features, steps, operations, elements and / orcomponents, but do not preclude the presence or addition of one or more other features, steps,operations, elements, components and / or groups thereof.The term 'further' is used in the embodiments and claims of the present application. The saidterm is a well-accepted term to narrow down any principal feature. Therefore, the person skilledin the art would clearly understand the scope of the said term in the context of the presentdisclosure.As used herein, the term 'reactive oxygen species (ROS)' refers to chemically reactive oxygencontaining molecules generated intracellularly, including but not limited to superoxide,hydrogen peroxide, and hydroxyl radicals, which contribute to oxidative stress.As used herein, the term 'IC50' refers to the concentration of the compound required to inhibit50% of cellular metabolic activity in vitro, as determined using assays such as MTT.As used herein, the term 'anticancer agent' refers to a compound capable of inhibitingproliferation or inducing death of cancer cells through mechanisms including but not limited toROS induction and mitochondrial disruption.The present invention is directed towards a Ruthenium (III) -based coordination complexhaving the structure formula IFormula Iwherein n - 9.In an embodiment of the present invention there is provided a Ruthenium (III)-basedcoordination complex, wherein the complex is trichlorido{6,6''-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2''-terpyridine]-κ3N}ruthenium(III).Another embodiment of the present invention provides a method for preparation of aRuthenium (III) -based coordination complex having the structure formula IFormula Iwherein n - 9,comprising the steps:a. dissolving a ruthennium salt in a first solvent to obtain a solution;b. dissolving a ligand in a second solvent to obtain a ligand solution;c. adding the solution dropwise to the ligand solution to obtain a reaction mixture;d. refluxing the reaction mixture at a temperature in the range of 60-70°C for 8-10hours; ande. removing the solvents to obtain the Ruthenium (III)-based coordination complex.In another embodiment of the present invention there is provided a method for preparation ofa Ruthenium (III)-based coordination complex, wherein the first solvent is ethanol.In yet another embodiment of the present invention there is provided a method for preparationof a Ruthenium (III)-based coordination complex, wherein the second solvent is chloroform.In still another embodiment of the present invention there is provided a method for preparationof a Ruthenium (III)-based coordination complex, wherein the ruthenium salt is Ruthenium(III)chloride.In an embodiment of the present invention there is provided a method for preparation of aRuthenium (III) -based coordination complex, wherein the ligand is 6,6"-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2"-terpyridine.In another embodiment of the present invention there is provided a method for preparation ofa Ruthenium (III) -based coordination complex, wherein the ruthenium salt and the ligand aretakeninaratioof1:1equivalentYet another embodiment of the present invention provides a method for preparation of aRuthenium (III) -based coordination complex having the structure formula IFormula Iwherein n - 9,comprising the steps:a. dissolving Ruthenium(III) chloride in ethanol to obtain a solution;b. dissolving a ligand in chloroform to obtain a ligand solution;c. adding the solution dropwise to the ligand solution to obtain a reaction mixture;d. refluxing the reaction mixture at a temperature of 65°C for 8 hours; ande. removing the solvents to obtain the Ruthenium (III)-based coordination complex.The present invention discloses a pyrene-terpyridine-polyethylene glycol-functionalizedruthenium(III) trichloride complex, herein designated as PTP-Ru, possessing a uniquemolecular architecture that integrates a pyrene moiety, a terpyridine coordinating ligand, and aPEG chain, within a single radiation-responsive platform. The ruthenium (III)-basedcoordination complex (PTP-Ru) is structurally engineered comprising (i) a pyrene moietycapable of efficient interaction with cellular components and radiation-induced excitation, (ii)a terpyridine ligand that stabilizes the ruthenium metal center and facilitates redox-mediatedbiological activity, and (iii) a polyethylene glycol (PEG) segment that improves aqueoussolubility, colloidal stability, and biocompatibility. This unique molecular architecture enablescontrolled activation of PTP-Ru in the presence of ionizing radiation, thereby enhancingtherapeutic selectivity.The different components of PTP-Ru and the function of each component is as following:1. Ruthenium (III) Center: Ruthenium metal ion (Ru3+) is coordinated via the terpyridineligand. The ion serves as the core radiation-responsive center. Upon exposure toionizing radiation (X-rays), the ruthenium center participates in redox reactions andfacilitates generation of reactive oxygen species (ROS), amplifying radiation-inducedcellular damage in cancer cells.2. Terpyridine Ligand: The Tridentate terpyridine moiety is coordinated to the rutheniumion. It provides structural stability to the ruthenium complex, tunes the electronicproperties of the metal center, and enables controlled redox activity necessary forradiation-induced activation.3. Pyrene Functional Group: The aromatic pyrene unit is conjugated to the terpyridineligand. It enhances cellular uptake and subcellular localization (especially nearmembranes and mitochondria), facilitates energy transfer upon radiation, andcontributes to enhanced ROS generation, which promotes cancer cell killing under Xray exposure.4. Polyethylene Glycol (PEG) Chain: The PEG chain is conjugated to the pyrene-terpyridine ligand. It improves aqueous solubility, biological stability, andbiocompatibility of the complex. It also reduces nonspecific toxicity, enhances systemicstability, and allows safe interaction with normal cells while maintaining selectiveradiosensitization of cancer cells.The ruthenium (III)-based coordination complex (PTP-Ru) is synthesized in good yield by thedropwise addition of RuCl3.XH2O dissolved in ethanol to a chloroform solution of the ligandPTP(tidilthllljt)idbttflkThtimixture is refluxed at 65°C for 8 h. Upon completion, the solvent is removed under reducedpressure, and the product is washed successively with ethanol and diethyl ether. Scheme 1below provide the process for synthesis of the PTP-Ru complex.Scheme 1The PTP-Ru complex is stable at room temperature and soluble in DMSO (1 mg / mL), CHCl3,CH2Cl2, CH3CN, C2H5OH and CH3OH. PEG conjugation ensures colloidal stability andimproved systemic delivery. The pyrene-terpyridine-PEG architecture allows PTP-Ru tofunction as a nanocarrier, combining: structural stability (terpyridine ligand); radiationresponsive activation (ruthenium center); cellular targeting and ROS facilitation (pyrene) andbiocompatibility and solubility (PEG).PTP-Ru exhibits enhanced biological activity specifically upon exposure to ionizing X-rayradiation, enabling controlled activation of the ruthenium center and distinguishing it fromconventional ruthenium complexes that functions independently of radiation. PTP-Ru providespreferential radiosensitization of cancer cells compared to normal cells under identical radiationconditions, establishing a favorable therapeutic window for radiation-assisted cancer therapy.This selectivity ensures preferential killing of cancer cells while minimizing damage to healthytissues. The inclusion of a polyethylene glycol (PEG) segment in PTP-Ru imparts improvedaqueous solubility, reduced nonspecific toxicity, and enhanced biocompatibility, which are notcommonly achieved simultaneously in existing ruthenium-based radiosensitizers. PEGylationof the complex enhances aqueous solubility, biocompatibility, and stability, reducingnonspecific cytotoxicity.The novelty of the present invention resides in the radiation-triggered dual validation of PTPRu, combining selective anticancer radiosensitization with demonstrated efficacy in a well-established radioresistant model system. Unlike conventional radiosensitizers or metal-basedtherapeutics, PTP-Ru offers controlled activation under X-ray irradiation, enhanced selectivity,and improved biocompatibility owing to its PEGylated design.The synthesized ruthenium complex PTP-Ru exhibits pronounced anticancer activity that issignificantly enhanced in the presence of ionizing radiation. The present invention is based onthe principle that PTP-Ru remains comparatively less cytotoxic in the absence of radiation,while undergoing radiation-triggered activation that amplifies cellular damage in cancer cells.The anticancer and radiosensitizing potential of PTP-Ru is evaluated using human cervicalcancer (HeLa) cells, a widely accepted in vitro model for studying radiation-mediated cancertherapy. HeLa cells are known to possess relatively robust survival mechanisms and thereforeserve as a suitable platform for assessing radiosensitizer efficacy. HeLa cells are treated withvarying concentrations of PTP-Ru and exposed to X-ray irradiation at a dose of 15 Gy. Cellviability is assessed under both irradiated and non-irradiated conditions. The resultsdemonstrate that PTP-Ru induces significantly higher cytotoxicity under X-ray exposure, withan observed IC50 value of approximately 0.27 mg / mL in irradiated cells. In contrast, PTP-Ruexhibits comparatively reduced cytotoxicity in the absence of radiation, confirming that thebiological activity of the complex is radiation-dependent. Non-irradiated cells showcomparatively lower cytotoxicity, confirming controlled, radiation-dependent activation. Theenhanced anticancer activity of PTP-Ru under irradiation is attributed to its ability to amplifyradiation induced oxidative stress, disrupt mitochondrial homeostasis, and initiate programmedcell death pathways. Without being bound by theory, it is believed that interaction of X rayswith the ruthenium center and pyrene moiety of PTP-Ru leads to increased production ofreactive oxygen species (ROS), thereby intensifying damage to critical cellular componentssuch as mitochondria, membranes, and nucleic acids. Through these mechanisms, PTP-Rufunctions as a radiosensitizer, amplifying radiation induced cellular damage withoutnecessitating an increase in radiation dose.Deinococcus radiodurans has been employed in the present invention as a radioresistantbiological model to validate the radiation sensitizing capability of PTP-Ru under extremeresistance conditions. Deinococcus radiodurans is widely recognized for its extraordinaryresistance to ionizing radiation, attributed to highly efficient DNA repair mechanisms, robustantioxidant defenses, and exceptional cellular recovery pathways. D. radiodurans cultures aretreated with PTP-Ru at a concentration of 1 mg / mL and subjected to X-ray irradiation at a doseof50Gy,aradiationlevelthattypicallydoesnotresultinsignificantlethalityinthisorganismunder normal conditions. The biological response is assessed using spot assay and spread plateassay methodologies. The results demonstrate a marked reduction in colony formation insamples exposed to the combined treatment of PTP-Ru and X-ray irradiation, compared to nonirradiated controls. These observations confirm that PTP-Ru enhances radiation inducedbiological damage even in a highly radioresistant system. Importantly, D. radiodurans is usedsolely as a radioresistance validation model, and no antimicrobial model. The ability of PTPRu to sensitize such a robust radioresistant model provides strong experimental support for itsmechanism as a radiation-amplifying agent, and underscores its potential effectiveness againstradioresistant cancer phenotypes. This indicates the ability of PTP-Ru to overcome extremeradiation resistance, providing strong proof-of-concept for treating radioresistant cancerphenotypes.Thus, the present invention provides PTP-Ru as a new class of radiation-activated rutheniumcomplexes suitable for use in cancer radiotherapy and radiosensitization strategies, withvalidated performance in both mammalian cancer models and radioresistant biological systems.The present invention fulfills a critical need for safer, more effective, and radiation-synergistictherapeutic agents in modern oncology. PTP-Ru remains largely inactive in the absence of Xray exposure, reducing unnecessary toxicity. Activation only upon irradiation allows for precisespatiotemporal control of the therapeutic effect, a significant advantage over conventionalchemotherapeutic agents.APPLICATIONS OF THE PRESENT INVENTIONThe present invention comprising the radiation-responsive ruthenium (III) -based coordinationcomplex (PTP-Ru) has multiple potential applications in the field of cancer therapy andradiation-assisted biomedical interventions. The utility of the PTP-Ru is derived from itsradiation-triggered anticancer activity, selective radiosensitization, and validated function inradioresistant biological models.- PTP-Ru can be employed as a radiosensitizing agent in radiotherapy protocols,particularly for solid tumors that exhibit partial resistance to conventional radiationtreatment.- The selective enhancement of radiation-induced cytotoxicity in cancer cells allows formore effective tumor control at lower radiation doses, potentially minimizing collateraldamage to surrounding healthy tissues.- PTP-Ru demonstrates efficacy in Deinococcus radiodurans as a radioresistant model,indicating its potential to overcome intrinsic or acquired radioresistance in tumor cells.PTP-Ru may thus be particularly useful in treating recurrent or resistant cancers, whichare often challenging to manage with conventional radiotherapy alone.- The nanocarrier architecture (PEGylated pyrene-terpyridine ruthenium complex)enables controlled, radiation-triggered activation, allowing precise spatiotemporaldelivery of therapeutic effects. This feature could be further exploited in combinationtherapies, where PTP-Ru is co administered with other chemotherapeutic or targetedagents to enhance overall efficacy.- PTP-Ru can serve as a tool for studying radiation-induced cellular processes,mitochondrial dysfunction, and ROS-mediated cytotoxicity in both cancer andradioresistant models.- PTP-Ru may also be used in high-throughput screening assays to identify novelradiosensitizers or to evaluate tumor radiosensitivity in vitro.- The structural design of PTP-Ru provides a template for further functionalization,including tumor-targeting ligands, imaging agents, or dual therapeutic modalities. Thismakes the PTP-Ru versatile for next-generation theranostic applications, combiningselective cancer therapy with diagnostic or imaging capabilities.EXAMPLESThe following examples are given by way of illustration, therefore, should not be construed tolimit the scope of the invention.EXAMPLE 1Synthesis of pyrene-terpyridine-polyethylene glycol-functionalized ruthenium(III)trichloride coordination complex [Trichlorido{6,6''-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2''-terpyridine]-κ3N}ruthenium(III)]RuCl3.XH2O (0.014 g, 0.067 mmol) was dissolved in ethanol (6 mL) to obtain a solution. Thissolution was added dropwise to a chloroform solution (6 mL) of the ligand PTP (pyrene-terpyridine-polyethylene glycol conjugate) [6,6"-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2"-terpyridine] (0.050 g, 0.04 mmol)] in a 50dbflkbiiihiiifld65°C for 8 h. Upon completion, the solvent was removed under reduced pressure, and theproduct (PTP-Ru complex) [Trichlorido{6,6''-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2''-terpyridine]-κ3N}ruthenium(III)] waswashed successively with ethanol and diethyl ether.EXAMPLE 2CHARACTERIZATIONThe formation of PTP-Ru complex was analyzed using UV, FL, and FT-IR.2.1 UV-vis AnalysisThe UV-visible spectrum of PTP-Ru (1.388 x 10-6 M) was recorded in CH3CN at 25 °C. Figure1(a) illustrates UV-vis absorption spectra of Ruthenium complex, PTP-Ru, in solution state at25°C. The spectrum exhibits absorption bands at 239, 274, 340, and 412 nm. The bands atlower wavelengths (239 and 274 nm) are assigned to π->π* transitions, while the band at 340nm is attributed to an n->π* transition. The absorption band at 412 nm is characteristic of ametal-to-ligand charge transfer (MLCT) transition.2.2 Photoluminescence StudiesFigure 1(b) illustrates normalised solution state fluorescence spectra of PTP-Ru at 25°C. Thesolution-state photoluminescence studies of PTP-Ru exhibit a single, sharp emission band witha maximum wavelength at λmax = 421 nm.Furthermore, the photoluminescence color of the complex was quantified using theCommission Internationale de l'Eclairage (CIE) chromaticity diagram. Figure 1(c) illustratesC.I.E. plot for PTP-Ru in solution state. PTP-Ru displays blue emission in the solution state,with CIE chromaticity coordinates of (x = 0.15659, y = 0.04363).2.3 FT-IR AnalysisFourier transform infrared spectroscopy (FT-IR) data was collected for (PTP-Ru) using aBruker Alpha-P Fourier Transform Infrared Spectrometer at 25°C (ATR method), shown inFigure 1(d).The FT-IR spectrum of PTP-Ru shows a broad -OH stretch at 3380 cm-1, aliphatic -CH2stretching at 2860 cm-1, aromatic C-H stretching at 3055 cm-1, an aromatic C=C stretch at1581 cm-1, and a strong PEG C-O-C stretch at 1045 cm-1, consistent with the successfulformation of the pyrene-terpyridine-PEG-Ru(III) complex.EXAMPLE 3Radiation-enhanced biological effects of PTP-Ru complex using spot assayDeinococcus radiodurans (procured from MTCC, Chandigarh, India; MTCC 4465) cultures of0.1 OD (overnight incubation) were treated with PTP-Ru at a concentration of 1 mg / mL. Thetreated bacterial samples, along with untreated (Control) were divided into two sets: one setwas exposed to X-ray irradiation of 50 Gy after 4 hours of incubation, while the other set waskept non-irradiated. 50 Gy is a radiation level that typically does not result in significantlethality in this organism under normal conditions. PTP-Ru was used at a concentration of 1mg / mL, from that the bacterial dilutions were prepared while keeping the PTP-Ruconcentration constant.Figure 2 illustrates the spot assay of Deinococcus radiodurans used as a radioresistant model,showing serial bacterial dilutions treated with PTP-Ru (1 mg / mL) in the absence and presenceof X-ray irradiation (50 Gy) to evaluate radiation-enhanced biological effects. The resultsdemonstrate a marked reduction in colony formation in samples exposed to the combinedtreatment of PTP-Ru and X-ray irradiation, compared to non-irradiated controls.EXAMPLE 4Radiation-enhanced biological effects of PTP-Ru complex using spread plate assayDeinococcus radiodurans (procured from MTCC, Chandigarh, India; MTCC 4465) cultures of0.1 OD (overnight incubation) were treated with PTP-Ru at a concentration of 1 mg / mL. Thetreated bacterial samples, along with untreated (Control) were divided into two sets: one setwas exposed to X-ray irradiation of 50 Gy after 4 hours of incubation, while the other set waskept non-irradiated. 50 Gy is a radiation level that typically does not result in significantlethality in this organism under normal conditions. PTP-Ru was used at a concentration of 1mg / mL, from that the bacterial dilutions (1 x 10-12) were prepared while keeping the PTP-Ruconcentration constant.Figure 3 illustrates the spread plate assay of Deinococcus radiodurans used as a radioresistantmodel, showing the effect of PTP-Ru (1 mg / mL) with and without X-ray irradiation (50 Gy)on colony formation. The figure 3 depicts a comparative bacterial survival assay performed onDeinococcus radiodurans using a high bacterial dilution (10-12) at a fixed concentration of 1mg / mL of PTP-Ru, evaluated in the presence and absence of X-ray irradiation (X-ray_Tp).1. Control: The untreated control plate shows a high density of bacterial colonies,confirming: excellent intrinsic survivability of D. radiodurans, successful plating at10-12 dilution, and no external stress affecting bacterial growth. This is consistent withthe well-known radioresistant and stress-tolerant nature of D. radiodurans.2. PTP-RuCl3 (1 mg / mL, No X-ray): The plate treated with PTP-Ru alone exhibitscomparable colony counts to the control, indicating: negligible inherent antibacterialtoxicity of PTP-Ru at 1 mg / mL, and good biocompatibility of PTP-Ru toward D.radiodurans in the absence of radiation. This suggests that PTP-Ru remains largelyinactive under normal physiological conditions.3. Control + X-ray_Tp: Exposure of D. radiodurans to X-ray irradiation alone results in:a moderate reduction in colony-forming units. However, substantial survival is stillobserved. This confirms that D. radiodurans retains strong intrinsic resistance toionizing radiation, even under X-ray exposure.4. PTP-Ru + X-ray_Tp: In sharp contrast, the combination of PTP-Ru (1 mg / mL) withX-ray irradiation leads to a drastic reduction in colony numbers, with only sparsesurviving colonies observed. This indicates synergistic interaction between PTP-Ruand X-ray radiation leading to effective radiation-mediated bacterial killing,overcoming the natural radioresistance of D. radiodurans. And activation of PTP-Ruspecifically under irradiation conditions. These results demonstrate a marked reductionin colony formation in samples exposed to the combined treatment of PTP-Ru and Xray irradiation, compared to non-irradiated controls.EXAMPLE 5BIOCOMPATIBILITY STUDIESC2C12 normal myoblast cells (procured from NCCS, Pune, Maharashtra, India) were culturedin DMEM supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in ahumidified 5% CO2 atmosphere. Cells were seeded in 96-well plates at a density ofapproximately 1 x 104 cells / well and allowed to attach for 24 h. PTP-Ru was then added atdifferent concentrations (0-0.5 mg / mL) [0.1, 0.2, 0.3, 0.4, 0.5 mg / mL] and incubated for 24 hat 37°C.For radiation-triggered biocompatibility assessment, treated cells were exposed to X-rayirradiation at a dose of 15 Gy (dose rate ~1 Gy / min) at room temperature (25 ± 2°C), followedby further incubation for 24 h at 37°C. Non-irradiated cells served as controls. Cell viabilitywas subsequently evaluated using the MTT assay, and results were expressed as percentageviability relative to untreated control cells.Figure 5 illustrates the biocompatibility and cytotoxicity assessment of PTP-Ru in C2C12normal cells, showing cell viability in the absence and presence of X-ray irradiation (15 Gy) toevaluate selective radiosensitization.In normal cells (C2C12 murine myoblasts), PTP-Ru exhibits a higher IC50 (~0.47 mg / mL)under identical irradiation conditions. This selectivity ensures preferential killing of cancercells while minimizing damage to healthy tissues. PEGylation of the complex enhancesaqueous solubility, biocompatibility, and stability, reducing nonspecific cytotoxicity.The biocompatibility evaluation in C2C12 normal cells demonstrates that PTP-Ru exhibits lowinherent cytotoxicity across the tested concentration range. Cell viability remained above theacceptablebiocompatibilitythreshold(~50%)upto~047mg / mLevenafterexposureto15Gy X-ray irradiation, indicating that normal cells largely tolerate both PTP-Ru and theradiation-triggered condition. The absence of a sharp viability drop at lower concentrationsconfirms that PTP-Ru does not induce significant off-target toxicity in normal cells. Overall,these results establish PTP-Ru as a biocompatible and radiation-safe system for normal tissues,supporting its suitability for selective, radiation-activated therapeutic applications.EXAMPLE 6CYTOTOXICITY AND ANTI-CANCER ACTIVITYHeLa (human cervical cancer cells) (procured from NCCS, Pune, Maharashtra, India) werecultured in DMEM (high glucose) supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified 5% CO2 atmosphere. Cells were seeded in 96-well platesat a density of approximately 1 x 104 cells / well and allowed to attach for 24 h at 37°C.PTP-Ru was dispersed in sterile PBS or serum-free DMEM and sonicated briefly (5-10 min)for uniform dispersion. PTP-Ru was tested at different concentrations (0-0.5 mg / mL) [0.1, 0.2,0.3, 0.4, 0.5 mg / mL]. Culture medium of the cells was replaced with PTP-Ru-containingmedium and cells were incubated with PTP-Ru for 24 h at 37°C. Untreated cells served ascontrols. After 24 h of PTP-Ru treatment, cells were divided into two groups:(a) Without X-ray: Plates were kept under standard incubation conditions.(b) With X-ray: Cells were exposed to X-ray irradiation at a dose of 15 Gy (at a dose rate of~1 Gy / min for ~15 min at room temperature (25 ± 2°C). Immediately after irradiation, plateswere returned to the incubator. Cells were then incubated for an additional 24 h at 37°C, 5%CO2 to allow radiation-induced effects to manifest.Cell viability was subsequently evaluated using the MTT assay. MTT reagent (5 mg / mL) wasadded at an amount of 10 μL / well and plates were incubated for 3-4 h at 37°C. Formazancrystals formed were dissolved in 100 μL DMSO and absorbance was measured at 570 nmusing a microplate reader. cell viability (%) was calculated relative to untreated controls.Experiments were performed in triplicate, and data were expressed as mean ± SD.Figure 4 illustrates the cytotoxicity evaluation of PTP-Ru in HeLa cancer cells, comparing cellviability in the absence and presence of X-ray irradiation (15 Gy) to demonstrate radiationenhanced anticancer activity.The results demonstrate that PTP-Ru induces significantly higher cytotoxicity under X-rayexposure, with an observed IC50 value of approximately 0.27 mg / mL in irradiated cells. Incontrast, PTP-Ru exhibited comparatively reduced cytotoxicity in the absence of radiation,confirming that the biological activity of the complex is radiation-dependent. The enhancedanticancer activity of PTP-Ru under irradiation is attributed to its ability to amplify radiationinduced oxidative stress, disrupt mitochondrial homeostasis, and initiate programmed cell deathpathways. Without being bound by theory, it is believed that interaction of X rays with theruthenium center and pyrene moiety of PTP-Ru leads to increased production of reactiveoxygen species (ROS), thereby intensifying damage to critical cellular components such asmitochondria, membranes, and nucleic acids.Although embodiments for the present subject matter have been described in languagespecific to features, it is to be understood that the present subject matter is not necessarilylimited to the specific features described. Rather, the specific features and methods aredisclosed as embodiments for the present subject matter. Numerous modifications andadaptations of the system / device of the present invention will be apparent to those skilled inthe art, and thus it is intended by the appended claims to cover all such modifications andadaptations which fall within the scope of the present subject matter.It will be further appreciated that functions or structures of a plurality of components or stepsmay be combined into a single component or step, or the functions or structures of one-step orcomponent may be split among plural steps or components. The present invention contemplatesall of these combinations. Unless stated otherwise, dimensions and geometries of the variousstructures depicted herein are not intended to be restrictive of the invention, and otherdimensions or geometries are possible. In addition, while a feature of the present invention mayhave been described in the context of only one of the illustrated embodiments, such feature maybe combined with one or more other features of other embodiments, for any given application.It will also be appreciated from the above that the fabrication of the unique structures hereinand the operation thereof also constitute methods in accordance with the present invention. Thepresent invention also encompasses intermediate and end products resulting from the practiceof the methods herein.

Claims

1. A Ruthenium (III) -based coordination complex having the structure formula I Formula I wherein n - 9.

2. The Ruthenium (III) -based coordination complex as claimed in claim 1, wherein the complex is trichlorido{6,6''-diyl-bis(oxy)-bis[3,6,9,12,15,18,21,24-octaoxahexacosan1-ol]-4'-(pyren-1-yl)-2,2':6',2''-terpyridine]-κ3N}ruthenium(III).

3. A method for preparation of a Ruthenium (III) -based coordination complex having the structure formula I Formula I wherein n - 9, comprising the steps: a. dissolving a ruthenium salt in a first solvent to obtain a solution; b. dissolving a ligand in a second solvent to obtain a ligand solution; c. adding the solution dropwise to the ligand solution to obtain a reaction mixture; d. refluxing the reaction mixture at a temperature in the range of 60-70°C for 8-10 hours; and e. removing the solvents to obtain the Ruthenium (III) -based coordination complex.

4. The method as claimed in claim 3, wherein the first solvent is ethanol.

5. The method as claimed in claim 3, wherein the second solvent is chloroform.

6. The method as claimed in claim 3, wherein the ruthenium salt is Ruthenium(III) chloride.

7. The method as claimed in claim 3, wherein the ligand is 6,6"-diyl-bis(oxy)- bis[3,6,9,12,15,18,21,24-octaoxahexacosan-1-ol]-4'-(pyren-1-yl)-2,2':6',2"- terpyridine.

8. The method as claimed in claim 3, wherein the ruthenium salt and the ligand are taken in a ratio of 1: 1 equivalent.