Encapsulation of a Ru(η⁶-p-cymene) complex of the antibacterial drug trimethoprim into a polydiacetylene-phospholipid assembly to enhance its in vitro anticancer and antibacterial activities

View Article Online View Journal NJC New Journal of Chemistry A journal for new directions in chemistry Accepted Manuscript This article can be cited before page numbers have been issued, to do this please use: D. Gopalakrishnan, C. sumithaa, M. Arumugam, N. Bhuvanesh, S. Ghorai, P. Das and G. Mani, New J. Chem., 2020, DOI: 10.1039/D0NJ03664A. This is an Accepted Manuscript, which has been through the Volume 42 Number 6 2 P 1 a M ge a s r c 3 h 9 2 6 0 3- 18 4776 Royal Society of Chemistry peer review process and has been accepted for publication. NJC Accepted Manuscripts are published online shortly after acceptance, New Journal of Chemistry A journal for new directions in chemistry rsc.li/njc before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. 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In no event ISSN 1144-2546 shall the Royal Society of Chemistry be held responsible for any errors PCYpH er Ah 2el O el P poc 2aw Eh r R a iie as ndh H d va u Kina , M dT a z n b u mO l-uJ 4iec e ra ln ou s wmL o i a ni x nv id ee es- t iza ca ise n l s .n g ist atgeg rde a n pht hysdenroet hoexridmea ql uroauntteu mus idnogt s or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains. rsc.li/njc Page 1 of 36 New Journal of Chemistry 1 2 3 Communicated to: New Journal of Chemistry 4 Manuscript type: Article 5 MS ID: NJ-ART-07-2020-003664 6 7 8 9 t 10 p Encapsulation of Ru(η6-p-cymene) complex of the antibacterial drug trimethoprim 11 i 12 into polydiacetylene-phospholipid assembly to enhance its In vitro anticancer and r c 13 antibacterial activities 14 s 15 u Durairaj Gopalakrishnan,a Chezhiyan Sumithaa,a Arumugam Madan Kumarb Nattamai S. P. 16 , n 17 Bhuvanesh,c Suvankar Ghorai,d Priyadip Dasa* and Mani Ganeshpandiana* a 18 M 19 20 d 21 22 aDepartment of Chemistry, SRM Institute of Science and Technology, Kattankulathur 603 203, e 23 Tamil Nadu, India t 24 bCancer Biology Lab, Molecular and Nanomedicine Research unit, Sathyabama Institute of p 25 Science and Technology, Chennai, Tamil Nadu, India. e 26 cX-ray Diffraction Lab, Department of Chemistry, Texas A&M University, College Station, TX c 27 c 77842, USA. 28 A 29 30 y 31 r 32 t 33 s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r u 47 48 o 49 J 50 51 w 52 e 53 N 54 55 56 57 *To whom correspondence should be addressed, e-mail: ganeshpm@srmist.edu.in and 58 priyadip@srmist.edu.in 59 60 1 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 2 of 36 1 2 3 4 5 Abstract 6 A new organometallic ruthenium(II)-p-cymene complex bearing the antibacterial drug 7 8 trimethoprim (RATMP(C)) has been synthesised, enwrapped into a polydiacetylene(PDA)- 9 based liposome (Lip-RATMP(C)), and studied for its anticancer and antibacterial activities. t 10 p 11 The conjugated yne-ene chain of polymeric backbone of PDA assists the formation of stable i 12 nanoaggregate from which only 50% of the encapsulated complex was leached out even after r c 13 110 h under physiological conditions. RATMP(C) shows DNA cleavage activity more efficient 14 s 15 than Lip-RATMP(C) and the DNA binding constant of complex determined using UV-Vis u 16 absorption spectral titration illustrating the role of hydrophobicity and hydrogen bonding n 17 propensity of coordinated ligands in determining the DNA binding affinity of the complex. a 18 M 19 Cell viability assay reveals that the toxicity of RATMP(C) in healthy HEK-293 cells could be 20 reduced when it is loaded into liposomes. Both RATMP(C) and Lip-RATMP(C) do not show d 21 marked cytotoxic activity against A549 human lung adenocarcinoma and MCF-7 human breast 22 e 23 carcinoma cells. But, Lip-RATMP(C) exhibits cytotoxic activity and apoptosis-inducing t p 24 ability against HepG2 human liver carcinoma cells with potency higher than that of complex 25 e illustrating the significance of formation of liposome-Ru(arene) complex nanoaggregate. In 26 c contrast, RATMP(C) showed potent antibacterial activity against Pseudomonas aeruginosa, 27 c 28 and Staphylococcus aureus, whereas Lip-RATMP(C) turned out to be inactive even in the A 29 higher concentration tested. 30 y 31 r 32 t 33 s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r u 47 48 o 49 J 50 51 w 52 e 53 N 54 55 56 57 58 59 60 2 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 3 of 36 New Journal of Chemistry 1 2 3 4 5 Introduction 6 7 The platinum(Pt)-based drugs such as cis-platin and its derivatives are clinically 8 9 used as the metal-based drugs for the treatment of different types of tumour t 10 p worldwide.1,2 However, the Pt-based drugs chaotically damage both cancerous and 11 i 12 r normal tissues after systemic administration and tumours frequently acquire resistance c 13 14 s to these drugs.3,4 In order to overcome the adverse effects of platinum-based anticancer 15 u 16 agents, the ruthenium(Ru)-based chemotherapeutic potency complexes like n 17 a 18 imidazolium trans-[tetrachlorido(dimethylsulfoxide)(1H-imidazole)ruthenate(III)] M 19 20 (NAMI-A), indazolium trans-[tetrachloridobis(1H-indazole)ruthenate(III)] (KP1019) d 21 22 and its sodium salt ((N)KP1339) have entered for clinical investigation studies for the e 23 t 24 treatment of cancer.5-7 Nowadays, the design of Ru-based metallopharmaceuticals p 25 e mainly focuses on the evaluation of anticancer activities of organometallic half- 26 c 27 c sandwich Ru(II)-arene complexes.8,9 For instance, Ru(II)-p-cymene-PTA complex 28 A 29 (RAPTA-C, PTA is 1,3,5-triaza-7-phosphaadamantane) considered as a leading 30 y 31 chemical structure to achieve clinical trials due to its anti-metastatic activity.10 r 32 t 33 Organometallic Ru(II)-arene complexes of the type [RuII(η6-arene)(YZ)(X)]+ (YZ = s 34 i m 35 bidentate ligand, X = halide) exhibited efficient cytotoxicity against different cancer 36 e 37 cells.11 h 38 C 39 It is well established that the incorporation of clinically-used drug into Ru(II)- 40 f 41 arene moiety confers the versatility on the structure of complexes, which can able to o 42 43 tune their anti-proliferative activity.12 Different category of drugs have been repurposed a l 44 n 45 for this idea, including quninolone antibacterial drugs,13 imidazole-containing 46 r u 47 antifungal drugs,14 Non-steroidal anti-inflammatory drugs,15 curcumin class of anti- 48 o 49 inflammatory drugs,16 chloroquine-based antimalarial drugs17 and antidiabetic drugs.18 J 50 51 Also, Ru(II)-arene complexes bearing a range of bioactive ligands including w 52 gluthathione-S-transferase inhibitor (ethacrynic acid),19a selective oestrogen receptor e 53 N 54 modulator (tamoxifen),19b protein kinase inhibitor (staurosporine),19c glycolysis 55 56 inhibitor (lonidamine),19d aromatase inhibitor (letrozole),19e cyclin-dependent kinase 57 58 inhibitor (Paullone),19f phenoxazine-containing multi-drug resistance inhibitor,19g and 59 60 3 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 4 of 36 1 2 3 naphthalimide-based DNA intercalator19h have been reported to exhibit anticancer 4 5 activity with new mechanisms of action. 6 7 8 In a related strategy, the combination of Ru-arene scaffold, which has great 9 interest in cancer metallotherapeutics, and a clinically-approved antibiotic drug, is t 10 p 11 reported to exhibit the dual activity, anti-microbial as well as anti-proliferative activity. i 12 r c 13 This strategy is desirable in clinics where the cancer patients are at a stage of potential 14 s 15 risk of infection due to the malignancy-related immunosuppression and/or specific u 16 n 17 defects include severe neutropenia, impaired neutrophil function, B-cell, T-cell, or NK- a 18 M 19 cell deficiency that happens during chemotherapy or radiation therapy.20 Sadler et al. 20 21 reported the dual functioning Ru(II)–p-cymene complex containing ciprofloxacin (anti- d 22 e 23 microbial drug) derivative for anti-proliferative and anti-bacterial activities.21a Elzbieta t p 24 Budzisz et al. reported the anticancer and anti-microbial properties of Ru(II)-η6-p- 25 e 26 c cymene complexes containing pyrazole carbothioamide derivatives.21b Inspired from 27 c 28 these, here in, we used a well-known antibacterial drug trimethoprim, as a coordinating A 29 30 ligand to prepare half-sandwich Ru(II)-p-cymene type complex [Ru(η6-p- y 31 r 32 cymene)(trimethoprim)Cl ] (RATMP-C). Trimethoprim is highly effective for the t 2 33 s 34 treatment of urinary tract infections (UTI).22 The antibacterial activity of trimethoprim i m 35 36 is mainly attributed to inhibit the bacterial dihydrofolate reductase (DHFR) with e 37 h million-fold more selectivity over mammalian DHFR, and disrupting the formation of 38 C 39 purines and thymidylate, which are involved in cell proliferation of bacteria.23 Therefore 40 f 41 the incorporation of trimethoprim into Ru(II)-arene moiety can lead to exhibit dual o 42 43 l pharmacological activity a 44 n 45 46 Although numerous ruthenium complexes have been found to exhibit potential r u 47 48 biological activity, the lack of biocompatibility as well as solubility and non-specific o 49 J side effects due to toxicity, limited their further development towards clinical 50 51 w applications.24 Liposomes, a nanoscale drug carrier, have addressed the current 52 e 53 challenges in the drug development process as they display excellent drug delivery N 54 55 efficiency, good biocompatibility, non-immunogenicity, bio-degradability, and long- 56 57 time blood circulation.25 Literature data reveals the advantages of liposomes as a drug 58 59 delivery cargo (i) to deliver hydrophilic, hydrophobic, and amphipathic nature of drugs 60 4 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 5 of 36 New Journal of Chemistry 1 2 3 (ii) to enhance the stability of drugs in physiological medium (iii) to avoid non-specific 4 5 interactions of drugs with biomolecules during administration (iv) to passively 6 7 extravasate and accumulate the drug in leaky vessels of tumours due to enhanced 8 9 permeability and retention (EPR) effect (iv) to achieve targeted delivery and controlled t 10 p release.26 Hence, liposomes are already progressing for delivering metal-based 11 i 12 r anticancer agents. For example, LipoplatinTM, which is a FDA-approved liposomal cis- c 13 14 s platin, is found to be accumulated in solid tumors 10- to 200-fold higher than that of 15 u 16 adjacent normal tissue.27 In this context, there are many encouraging reports describing n 17 a 18 the enhancement of anticancer activity of liposome-encapsulated ruthenium complex M 19 20 compared with the non-encapsulated complex.28 The liposome-encapsulated Ru- d 21 22 polypyridyl complex of the type [Ru(phen) (dppz)](ClO ) , where phen is 1,10- e 2 4 2 23 t 24 phenanthroline and dppz is dipyrido[3,2-a:2′,3′-c]phenazine, is reported as a theranostic p 25 e agent for triple-negative breast cancer cells.29 Chen et al reported the loading of 26 c 27 c ruthenium complex [Ru(ttbpy) PIP]2+ (ttby = 4,4’-ditertiarybutyl-2,2’-bipyridine; PIP = 28 2 A 29 2-phenyl-1H-imidazo-[4,5-f]-[1,10]phenanthroline) into pegylated liposomes to 30 y 31 improve its anticancer activity against human cervical cancer cell lines via r 32 t 33 mitochondrial pathway.30a In addition to improve the anticancer activity, liposomal s 34 i m 35 formulation was able to significantly enhance the antimicrobial activity compared with 36 e 37 the drug alone.30b The antibacterial drug gentamicin, after being encapsulated into h 38 liposome, exhibited significant antibacterial activity against highly gentamicin-resistant C 39 40 mucoid and non-mucoid clinical strains of P. aeruginosa.30c This is because the lipid f 41 o 42 bilayer of liposomes is able to fuse with the outer membrane of bacteria, leading to 43 l a 44 overcome the bacterial resistance and to improve the therapeutic index of a drug. Thus, n 45 46 liposomal formulation of drug could be developed as a better drug candidate for r u 47 48 obtaining improved pharmacological activities. But still the evaluation of o 49 J 50 pharmacological activity of liposome-organometallic Ru(arene) complex 51 w 52 nanoaggregate has remained scarce in literature. e 53 N 54 The primary aim of the present study is twofold. Firstly, we tethered the 55 56 antibiotic drug trimethoprim into Ru(II)-p-cymene scaffold with an intention to achieve 57 58 anticancer as well as antimicrobial activities. Secondly, the synthesized complex was 59 60 enwrapped into a lipid based self-assembled liposomes for further improvement of its 5 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 6 of 36 1 2 3 pharmacological activities. The another reason for encapsulation of complex into 4 5 liposome is the administration of complex in the free form would seriously limit its 6 7 further application, due to toxicity of metal complex, especially In vivo. For these 8 9 purposes, a polydiacetylene (PDA)-based liposome (Lip-RATMP(C)), which was t 10 p composed of two different components such as 10,12-pentacosadiynoic acid (PCDA) 11 i 12 r and a phospholipid, 1,2-ditetradecanoyl-sn-glycero-3-phosphocholine (DMPC), was c 13 14 s prepared (Scheme 1).The PDA-based liposome was utilized owing to its very unique 15 u 16 electrical and optical properties.31 The co-assembly of PDAs together with DMPC n 17 a 18 phospholipid has the advantage to tune the size, surface charge, stability of polymeric M 19 20 backbone PCDA. Lip-RATMP(C) was characterised by UV-Vis absorption d 21 22 spectroscopy, Transmission Electron Microscopy (TEM), Scanning Electron e 23 t 24 Microscopy (SEM), energy-dispersive X-ray analysis (EDX), dynamic light scattering p 25 e (DLS) and zeta potential measurements. The anti-proliferative and anti-microbial 26 c 27 c activities of Lip-RATMP(C) were analyzed and compared with RATMP(C). The results 28 A 29 indicated that the nano-aggregate could be a better anticancer drug candidate than a 30 y 31 non-encapsulated complex. However, it was found to be ineffective to enhance the r 32 t 33 antibacterial activity of the complex. s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r 47 u 48 o 49 J 50 51 w 52 e 53 N 54 Scheme 1. Schematic illustration of Lip-RATMP(C) nanoaggregate designed. 55 56 57 Materials and Methods 58 59 Materials 60 6 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 7 of 36 New Journal of Chemistry 1 2 3 [(ƞ6-p-cymene)RuCl ] precursor, trimethoprim, calf thymus (CT) DNA (highly 2 2 4 5 polymerized), pBR322 supercoiled DNA, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 6 7 2H-tetrazolium bromide (MTT) and 10,12-pentacosadiynoic acid (PCDA) were 8 9 obtained from Sigma-Aldrich (USA). 1,2-ditetradecanoyl-sn-glycero-3- t 10 p phosphocholine (DMPC) was obtained from Avanti Polar Lipids (Alabaster, AL). 11 i 12 r Ultrapure MilliQ water was used in all experiments. Phosphate buffered saline (PBS) c 13 14 s (pH 7.2) solution and 5 mM Tris HCl/50 mM NaCl buffer were prepared by the reported 15 u 16 procedure. Commercial solvents were distilled and then used for preparation of n 17 a 18 complexes. M 19 20 21 Preparation of [Ru(ƞ6-p-cymene)(trimethoprim)Cl ] (RATMP(C)) d 2 22 e 23 RATMP(C) was prepared by adding a methanolic solution (20 ml) of trimethoprim t p 24 (0.582g, 2 mmol) to the solution of the ruthenium dimer precursor [(ƞ6-p- 25 e 26 c cymene)RuCl ] (0.61g, 1mmol) in methanol (10 ml) and then the mixture was stirred 27 2 2 c 28 at 40 °C for 5 h. The yellow solid obtained was filtered and the product was separated A 29 30 by using suction filtration, washed twice with cold methanol and diethyl ether, and then y 31 r 32 the product was dried under vacuum. The yellow coloured crystals of complex, which t 33 s 34 are suitable for X-ray diffraction studies, were obtained by dissolving the complex in i m 35 36 acetonitrile and allowing it for crystallization using ether diffusion method. Yield: 79%; e 37 h CHN analysis for [Ru(η6-p-cymene)(trimethoprim)Cl ] Calculated: C, 48.32; H, 5.41; 38 2 C 39 N, 9.39. Found: C, 48.41; H, 5.36; N, 9.28 %. ESI-MS: [Ru(η6-p- 40 f 41 cymene)(trimethoprim)(H O)Cl]+ displays a peak at m/z 579 (calcd 579.13).1H NMR o 42 2 43 l (500 MHz, CDCl 3 ): δ 1.21-1.23 (d, 6H, CH(CH 3 ) 2 ), 1.95 (s, 3H, CH 3 ), 2.93 – 2.95 (m, a 44 n 45 1H, CH(CH ) ), 3.39 (s, 2H, CH ), 3.74 (s, 3H, O-CH ), 3.76 (s, 6H, O-CH ), 5.31 (d, 3 2 2 3 3 46 r u 47 4H, η6-C H ), 5.62 (s, 2H, NH ), 6.30-8.01 (s, 3H, aromatic). UV–Vis. (λ nm, (ε M−1 6 4 2 max 48 o 49 cm−1)): 289 (39,584), 421 (4072). J 50 51 w 52 e Preparation of Lip-RATMP(C) 53 N 54 Suitably weighted PCDA and DMPC were dissolved in CHCl at 4:1 molar ratio in a 55 3 56 round bottom flask and mixed with a acetonitrile solution of RATMP(C) complex 57 58 (stock solution concentration = 2mg/2ml). A milky white layer was obtained by solvent 59 60 evaporation using a rotary evaporator at 45 °C. The white layer obtained was hydrated 7 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 8 of 36 1 2 3 with 10 mM PBS (pH 7.2) and mixed with a vortex mixer. Then the resulting aqueous 4 5 mixture was sonicated for 30 minutes at 75 °C – 80 °C. After sonication, the hot milky 6 7 white semi-transparent liposome solution was filtered using a 0.4 µm syringe filter. The 8 9 filtrate was cooled to room temperature and stored overnight at 4 °C. Non-encapsulated t 10 p RATMP(C) complex has been removed from the obtained solution by centrifugation 11 i 12 r and the assemblies were re-dispersed in 10 mM PBS (pH = 7.2) solution. The samples c 13 14 s were irradiated using a UV lamp (254 nm, 400 μW/cm2, Luzchem photo reactor) for 30 15 u 16 minutes to get the polymerized PCDA/DMPC vesicles solution, which is blue in colour n 17 a 18 and then stored at 4 oC. The stability of the liposomes was monitored by recording the M 19 20 nature of the UV-Vis absorption spectrum for 15 days. At the beginning, the absorption d 21 22 spectra was recorded and then the stability was monitored 24 hours time interval up to e 23 t 24 5 days followed by 48 hours time interval up to 15 days. The bare liposome without p 25 e complex encapsulation was also prepared following the same procedure as described 26 c 27 c above. 28 A 29 30 y 31 r 32 Solid-state structure determination using X-ray Crystallography t 33 s 34 A BRUKER Quest X-ray (fixed-Chi geometry) diffractometer was employed for i m 35 36 crystal screening, unit cell determination and data collection of complex.32-35 Leica MZ e 37 h 75 microscope was used to identify a suitable yellow block with very well defined faces 38 C 39 from a representative sample of crystals of the same habit. The crystal ofsuitable size 40 f 41 selected was mounted on a nylon loop and then placed in a cold nitrogen stream o 42 43 l (Oxford) maintained at 100 K. The goniometer was controlled using the APEX3 a 44 n 45 software suite.32 The sample was optically centered with the aid of a video camera such 46 r u 47 that no translations were observed as the crystal was rotated through all positions. The 48 o 49 detector was set at 3.4 cm from the crystal sample. The X-ray radiation employed was J 50 51 generated from a Mo-Iμs X-ray tube (K  = 0.71073Å). 45 data frames were taken at w 52 e 53 widths of 1. These reflections were used to determine the unit cell. The unit cell was N 54 verified by examination of the h k l overlays on several frames of data. No super-cell or 55 56 erroneous reflections were observed. After careful examination of the unit cell, an 57 58 extended data collection procedure (4 sets) was initiated using omega scans. 59 60 8 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 9 of 36 New Journal of Chemistry 1 2 3 The integrated intensity information for each reflection was obtained by 4 5 reduction of the data frames with the program APEX3. The integration method 6 7 employed a three dimensional profiling algorithm and all data were corrected for 8 9 Lorentz and polarization factors, as well as for crystal decay effects. Finally the data t 10 p was merged and scaled to produce a suitable data set. The absorption correction 11 i 12 r program SADABS was employed to correct the data for absorption effects. Systematic c 13 14 s reflection conditions and statistical tests of the data suggested the space group P-1. A 15 u 16 solution was obtained readily (Z=4;Z'=2) using XT/XS in APEX3. Hydrogen atoms n 17 a 18 were placed in idealized positions and were set riding on the respective parent atoms. M 19 20 All non-hydrogen atoms were refined with anisotropic thermal parameters. Absence of d 21 22 additional symmetry and voids were confirmed using PLATON (ADDSYM). The e 23 t 24 structure was refined (weighted least squares refinement on F2) to convergence. p 25 e 26 c 27 c 28 Calculation of encapsulation efficiency A 29 30 Lip-RATMP(C) vesicles were prepared as reported above, and left to settle down y 31 r 32 overnight at 4 ◦C. Then the unencapsulated RATMP(C) complex has been separated t 33 s 34 from the resulting solution by centrifugation and the emission intensity was measured. i m 35 36 The complex encapsulation efficiency (EE), which is correlated with the concentration e 37 of the RATMP(C) that was not incorporated, can be expressed by equation (1).36 As the h 38 C 39 concentration of the complex is directly proportional to the emission intensity, the 40 f 41 equation 1 can be expressed as equation 2. The emission intensity of the complex o 42 43 l incorporated in liposome is equal to the emission intensity of the complex that was not a 44 n 45 incorporated subtracted from the total emission intensity of the complex before 46 r u 47 encapsulation; EE can be calculated using equation (3):37 The encapsulation efficiency 48 o 49 of RATMP(C) into liposomes was calculated as 51.73%. J 50 51 w 𝐴𝑐𝑡𝑢𝑎𝑙 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑎𝑟𝑡𝑒𝑑 𝑖𝑛 𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠 52 𝐸𝐸 =  100% (1) e 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑡ℎ𝑒𝑜𝑟𝑦 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑙𝑜𝑎𝑑𝑒𝑑 𝑖𝑛 𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠 53 N 54 𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑠𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑎𝑟𝑡𝑒𝑑 𝑖𝑛 𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠 55 𝐸𝐸 =  100% (2) 𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑦𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑡ℎ𝑒𝑜𝑟𝑦 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑙𝑜𝑎𝑑𝑒𝑑 𝑖𝑛 𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠 56 57 𝐸𝐸= 𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑡ℎ𝑒𝑜𝑟𝑦 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑙𝑜𝑎𝑑𝑒𝑑  𝐸𝑚𝑖𝑠𝑠𝑖𝑜𝑛 𝑖𝑛𝑡𝑒𝑛𝑠𝑖𝑡𝑦 𝑜𝑓 𝑡ℎ𝑒 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑛𝑜𝑡 𝑖𝑛𝑐𝑜𝑟𝑝𝑜𝑟𝑎𝑡𝑒𝑑  100% (3) 58 𝐶𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑡ℎ𝑒𝑜𝑟𝑦 𝑎𝑚𝑜𝑢𝑛𝑡 𝑜𝑓 𝑅𝐴𝑇𝑀𝑃(𝐶) 𝑙𝑜𝑎𝑑𝑒𝑑 𝑖𝑛 𝑙𝑖𝑝𝑜𝑠𝑜𝑚𝑒𝑠 59 60 9 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 10 of 36 1 2 3 4 5 6 In vitro Release Profile 7 8 The release profile of RATMP(C) from Lip-RATMP(C) was studied using the 9 t 10 p dialysis method. Lip-RATMP(C) suspension (2 ml) was transferred into a dialysis bag 11 i 12 r (MWCO 3 kDa) and the bag was dipped in 40 ml of 5% DMSO/10 mM PBS (pH = 7.2) c 13 14 solution at 37 °C. The aliquot of the release medium (2 ml) was collected and replaced s 15 u 16 with an equal amount of original PBS solution at different time intervals for 10 days. n 17 a 18 The emission spectra were recorded from 300 nm to 500 nm for the emission intensity M 19 20 vs. time plot ( = 284 nm and  = 345 nm) using a fluorescence spectrophotometer. Ext Mon d 21 22 The amount of RATMP(C), which was released from Lip-RATMP(C), was measured e 23 t from the emission intensity and the results are presented as mean ± s.d. of triplicates. p 24 25 e 26 c 27 c DNA Binding and DNA cleavage Studies 28 A 29 The DNA binding and DNA cleavage studies were carried out by using the 30 y 31 procedures reported in literature38 and the details are provided in supporting r 32 t 33 information. s 34 i m 35 Cell culture and proliferation assay 36 e 37 h The anti-proliferative activity of the compounds was studied by adopting MTT 38 C 39 proliferation assay. HEK-293 human embryonic kidney cells, A549 human lung 40 f 41 adenocarcinoma cells, MCF-7 human breast carcinoma cells and HepG2 human liver o 42 43 carcinoma cells were procured from National Centre for Cell Science (NCCS), Pune. l a 44 n 45 The cells in monolayer culture were cultivated in Dulbecco’s modified Eagle’s medium 46 r u 47 (DMEM) culture media supplemented with 10% heat-inactivated fetal bovine serum, 48 o 49 penicillin (100 U/ml) and streptomycin (100 μg/ml) (Invitrogen Corporation, CA, J 50 51 USA). Cells were trypsinized and plated at a density of ~ 20, 000 cells/well in 96-well w 52 plate and incubated in 5% CO at 37 °C in the CO incubator. The stock solutions of the e 53 2 2 N 54 compounds were prepared in 5% DMSO/10 mM PBS mixture immediately prior to 55 56 dilution. Different concentrations of solution were prepared by the dilution of the stock 57 58 solution using culture media without delay. The solutions were added in triplicate at the 59 60 appropriate concentrations in the respective wells. The final DMSO concentration in 10 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 11 of 36 New Journal of Chemistry 1 2 3 the wells did not exceed 0.2% and the same amount of DMSO was maintained in all the 4 5 cellular experiments. Therefore, all the experiments were done at non-toxic 6 7 concentrations of DMSO. Cells treated with 20 μM quercetin for 24 h were considered 8 9 as a positive control, while the untreated cells incubated with 0.2% DMSO for 24 h at t 10 p 37 °C were considered as a negative control. After treatment period, the medium was 11 i 12 r removed and 20 µL of MTT (5 mg/ml) was added to the cells and incubated at 37 °C c 13 14 s for 4 h. The MTT insoluble formazan was dissolved in DMSO and the MTT reduction 15 u 16 was quantified by measuring the absorbance at 570 nm (Multiskan Spectrophotometer, n 17 a 18 USA). The obtained data were plotted and fitted using GraphPad Prism software to M 19 20 calculate the 50% viability (IC ) value. The data were obtained for three biological 50 d 21 22 replicates each and used to calculate the mean. The IC values provided are mean ± e 50 23 t 24 standard deviation. The statistical significance (p-value) of the data, which was p 25 e determined using GraphPad prism software with t-test, is > 0.002 to < 0.05. 26 c 27 c Prior to the experiment, the stability of complex in 5% DMSO/10 mM PBS was 28 A 29 monitored by recording the UV-Vis absorption spectra of complex in different time 30 y 31 interval (Figure S1) in which no significant changes observed in the spectral pattern up r 32 t 33 to 24 hours. Since 5% DMSO/10 mM PBS mixture was used to prepare the initial stock s 34 i m 35 solution to carry out the biological studies, we have recorded the 1H-NMR spectra of 36 e 37 the RATMP(C) in DMSO-d6 medium (see details in Supporting Information, Figure h 38 S2). This spectrum is similar to the spectrum obtained in CDCl medium (Figure S3). C 39 3 40 We have also recorded the emission spectrum of RATMP(C) in 5% DMSO/10 mM PBS f 41 o 42 (stock solution) diluted with an appropriate volume of culture media and the emission 43 l a 44 spectral nature is similar to that of emission spectrum obtained in complete 5% n 45 46 DMSO/10 mM PBS medium (Figure S4). These results clearly assured the r u 47 48 considerable stability of the complex in the DMSO as well as medium used for MTT o 49 J 50 assay. Furthermore, the release profile of liposome-encapsulated complex in 5% 51 w 52 DMSO/10 mM PBS (stock solution) and using cell culture media outside the dialysis e 53 bag, instead of PBS was carried out as a control experiment to assess the expected N 54 55 release of the complex in the time frame of the MTT assay by measuring the emission 56 57 intensity of cell culture media outside the dialysis bag (Figure S5). 58 59 60 11 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 12 of 36 1 2 3 Acridine Orange (AO)/ Ethidium Bromide (EB) staining assay 4 5 AO/EB staining was used to evaluate cell and nuclear morphology as described 6 7 by the previous method.39,40 HepG2 human Liver cancer cell line for 24 hrs was seeded 8 9 on 24 well plates with 10% of FBS medium, around 50,000 cells in each well. The t 10 p apoptotic assay was performed after treatment with complex and nanoconjugate for 24 11 i 12 r hrs. AO is a nuclei stain, it will stain both live and dead cells, and EB will penetrate c 13 14 s stain the dead cells. 10 µg/ml of both AO/EB was processed with 5 minutes incubation. 15 u 16 Coverslips were taken, kept on glass slides and stained with 100 μL of the dye mixture n 17 a 18 (1 : 1 ratio of AO and EB), and it was immediately viewed under an inverted M 19 20 fluorescence microscope (EVOS FL digital inverted fluorescence microscope (AMG)). d 21 22 The result was recorded with the Green, yellow, orange and red fluorescent emitting e 23 t 24 cells based on the live, early apoptosis, late apoptosis and necrosis cells respectively. p 25 e 26 c 27 c Antibacterial activity assay 28 A 29 The antibacterial activity of RATMP(C), Lip-RATMP(C) and trimethoprim was 30 y 31 tested on the Gram-positive Staphylococcus aureus (S. aureus) and Gram- r 32 t 33 negative Pseudomonas aeruginosa (P. aeruginosa) bacterial strains using the agar disk s 34 i m 35 diffusion method as reported in literature.41,42 Bacteria were allowed to grow on Luria- 36 e 37 Bertani medium supplemented with agar (Hi Media Laboratories). The compounds h 38 were applied at a concentration of 25, 50, 75, and 100 μM accordingly on to the paper C 39 40 disk (6 mm diameter). The sample-treated disks were carefully placed onto the pre- f 41 o 42 spread plates of bacterial strains. All these plates were incubated at 37 °C for 24 h and 43 l a 44 the zone of inhibition (mm) was measured. The data were obtained for three replicates n 45 46 each and expressed in means ± SD. r u 47 48 o 49 J 50 51 w 52 e 53 N 54 55 56 Results & Discussion 57 58 Synthesis and Characterization of RATMP(C) 59 60 12 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 13 of 36 New Journal of Chemistry 1 2 3 The method of preparation of RATMP(C) is provided in section 2.2. Briefly, the 4 5 Ru-p-cymene dimeric precursor complex is mixed with trimethoprim ligand in 2:1 6 7 molar ratio and stirred for 5 h in methanol and the desired complex was obtained as a 8 9 yellow-colour solid by filtration. The isolated complex is soluble in chloroform, t 10 p methanol, acetonitrile and DMSO solvents. The solution-state structure of complex was 11 i 12 r characterized by conventional spectroscopic techniques and solid-state structure of the c 13 14 s complex was elucidated by X-ray crystallography technique. The mass spectrum of the 15 u 16 complex displays m/z peak corresponding to [Ru(η6-p- n 17 a 18 cymene)(trimethoprim)(H O)Cl]+, which confirms the formation of complex and its 2 M 19 20 identity in solution state (Figure S6). The molecular formula of the complex was d 21 22 confirmed by structural elucidation of [Ru(η6-p-cymene)(trimethoprim)Cl ] (Figure 1) e 2 23 t 24 using X-ray crystallography (cf. below). p 25 e 26 c 27 c 28 A 29 30 y 31 r 32 t 33 s 34 i m 35 36 Ru e 37 h 38 N1 C 39 Cl1 40 Cl2 f 41 o 42 43 l a 44 n 45 46 Figure 1. ORTEP representation of the crystal structure of [Ru(η6-p- r u 47 cymene)(trimethoprim)Cl ] (RATMP-C). (hydrogen atoms are omitted for clarity) 48 2 o 49 J 50 51 w The UV-Vis absorption spectrum of complex shows a high intense band at 289 52 e 53 nm corresponding to intra-ligand π–π* transitions and a weak absorption band at 421 N 54 55 nm due to metal-to-ligand charge transfer (MLCT) transition (Figure 2a). In the 1H 56 57 NMR spectrum of complex (Figure S3), the characteristic six –CH(CH ) protons and 3 2 58 59 three –CH protons of the p-cymene ligand have appeared as a doublet (δ 1.21 ppm) 3 60 13 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 14 of 36 1 2 3 and singlet (δ 1.95 ppm) respectively. The septet signal, which is corresponding to 4 5 CH(CH ) proton appears in the region at (δ) 2.93 – 2.95 ppm. The nine O-CH protons 3 2 3 6 7 of the trimethoprim ligand are observed as two singlet at 3.74 and 3.76 ppm. The signal 8 9 due to –NH 2 protons of trimethoprim ligand, which appears in the region as a singlet at t 10 p 5.62 ppm, is observed as a broad peak presumably because of hydrogen bonding 11 i 12 r interaction. c 13 14 s Description of Crystal Structure of RATMP(C). Suitable crystals of the complex for 15 u 16 X-ray diffraction were obtained from the saturated acetonitrile solution of the complex n 17 a 18 using the ether diffusion method. The crystallographic data and selected bond lengths M 19 20 and bond angles of the complex are listed in Tables 1 and 2 respectively. Results of d 21 22 structure refinement reveal that the complex crystallizes in the triclinic crystal system e 23 t 24 with P1space group and the asymmetric unit of complex contains a neutral molecule. p 25 e The complex adopts typical ‘‘three-legged piano-stool” geometry in which Ru(II) metal 26 c 27 c centre is coordinated with six C-atoms of the p-cymene ring, nitrogen atom (N1) of 28 A 29 pyrimidine group, and two chlorido ligands (Cl1, Cl2) in pseudo-octahedral fashion 30 y 31 (Figure 1).43,44 The bond angles made by N1 atom of trimethoprim and chlorido ligands r 32 t 33 are nearly about 90o (N(1)–Ru(1)–Cl(1), 86.13(5)o; N(1)–Ru(1)–Cl(2), 89.63(5)o; s 34 i m 35 Cl(1)–Ru(1)–Cl(2), 87.11(2)o), which confirms the ‘piano-stool’ structure of the 36 e 37 complex. The selected bond distances, for instance, Ru(1)-(N1) (2.147(18) Å), Ru(1)- h 38 (Cl1) (2.415(6) Å), Ru(1)-(Cl2) (2.434(6) Å) and Ru-Cent (1.646(5) Å), are comparable C 39 40 to those reported for the similar analogue of half-sandwich Ru(II)-p-cymene f 41 o 42 complexes.11c The dihedral angle between the plane of 2,4-diamino-5-pyrimidine and 43 l a 44 3,4,5-trimethoxybenzyl ring is 114.30(19)o, revealing that the later scaffold is non- n 45 46 planar with regard to the pyrimidine moiety. Interestingly, 2-amino group of r u 47 48 trimethoprim ligand involves inter-molecular (N(4)-H(4b)...Cl(2): 2.643 Å) and intra- o 49 J 50 molecular (C(5b)-H(5ba)...N(4): 2.683 Å) hydrogen bonding interaction whereas 4- 51 w 52 amino group involves two intra-molecular hydrogen bonding interactions (N(3)- e 53 H(3a)...Cl(2b): 2.610 Å; N(3)-H(3b)...Cl(1b): 2.648 Å). Further, chlorido ligand Cl(1) N 54 55 involves inter-molecular (C(22)-H(22c)...Cl(1): 3.000 Å) and intra-molecular (N(3b)- 56 57 H(3ba)...Cl(1): 2.731 Å) hydrogen bonding interactions (Figure S7). 58 59 60 14 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Table 1. Crystal data and structure refinement details for RATMP(C). Formula C H Cl N O Ru 24 32 2 4 3 Formula weight 596.50 Crystal System Triclinic Space group P -1 a, Å 12.2293(10) b, Å 15.4509(13) c, Å 15.9391(13) , deg 67.587(3) , deg 70.606(3) , deg 77.965(3) Volume [Å] 2614.6(4) Z 4 D(calc) [mg/m3] 1.515 Goodness-of-fit on F 2 1.114 Final R indices R = 0.0310, wR2 = 0.0639 1 R indices (all data) R = 0.0402, wR2 = 0.0684 1 Table 2. Selected bond distances [Å] and bond angles [deg] for RATMP(C). Bond Distance [Å] Ru(1)-N(1) 2.147(18) Ru(1)-Cl(1) 2.415(6) Ru(1)-Cl(2) 2.434(6) Ru(1)-Cent 1.646 (5) Bond Angle [deg] N(1)-Ru(1)-Cl(1) 86.13(5) N(1)-Ru(1)-Cl(2) 89.63(5) Cl(1)-Ru(1)-Cl(2) 87.11(2) 0.9 0.6 0.3 0.0 200 400 600 800 1000 1200 15 ecanbrosbA 200.0k (a) RATMP(C) Lip-RATMP(C) Lip 0.0 450 500 550 600 650 700 750 800 Wavelength(nm) )u.a( ytisnetnI ecnecseroulF Page 15 of 36 New Journal of Chemistry 1 2 3 4 5 6 7 8 9 t 10 p 11 i 12 r c 13 14 s 15 u 16 n 17 a 18 M 19 20 d 21 22 e 23 t p 24 25 e 26 c 27 c 28 A 29 30 y 31 r 32 t 33 s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r (b) u 47 48 o 49 J 50 51 w 52 Lip-RATMP(C) e 53 N 54 55 56 57 RATMP(C) 58 59 60 Wavelength (nm) .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A (c) (e) (g) 16 2 0 0 n m New Journal of Chemistry Page 16 of 36 1 2 3 4 5 6 7 8 9 t 10 p 11 i 12 r c 13 14 s (d) 15 u 16 n 17 a 18 M 19 20 d 21 22 e 23 t p 24 25 e 26 c 27 c 28 A 29 30 y 31 r 32 (f) t 33 s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r u 47 48 o 49 J 50 51 w 52 e 53 N 54 55 56 57 58 59 60 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 17 of 36 New Journal of Chemistry 1 2 3 4 5 6 7 8 9 t 10 p 11 i 12 Figure 2. Characterization of Lip-RATMP(C). (a) UV-Vis absorption spectra of RATMP(C) r c 13 (acetonitrile), bare liposome (10 mM PBS, pH 7.2) and Lip-RATMP(C) (5% acetonitrile/10 14 s mM PBS, pH 7.2) (b) Fluorescence spectra of RATMP(C) and Lip-RATMP(C) (5% 15 u 16 acetonitrile/10 mM PBS, pH 7.2,  ex = 420 nm,  em = 521 nm). Morphological observation n 17 for Lip-RATMP(C) by using (c) TEM and (d) SEM. EDX analysis of Lip-RATMP(C) using a 18 (e) TEM and (f) SEM. (g) Particle size distribution of Lip-RATMP(C) measured by DLS. M 19 20 d 21 22 e 23 t Synthesis and Characterization of Lip-RATMP(C) p 24 25 e The isolated RATMP(C) complex was loaded into liposomes, which comprise 26 c 27 c of PCDA and one phospholipid, namely DMPC in a certain molar ratio of (4:1) using 28 A 29 bio-molecular self-assembly. The obtained liposomal solution was purified and 30 y 31 irradiated with UV light (= 254 nm) to get Lip-RATMP(C) with the encapsulation r 32 t 33 efficiency of 51.73%. The noticeable colour change from colourless to blue confirmed s 34 i m 35 the polymerization process occurred.45 The UV-Vis absorption spectrum of 36 e 37 nanoaggregate shows the characteristic absorption band at 652 nm illustrating the h 38 39 presence of stable ene-yene-conjugated polymeric backbone in the PCDA/DMPC C 40 vesicles (Figure 2a).46 Moreover, no apparent changes observed in the electronic f 41 o 42 absorption spectrum of Lip-RATMP(C) in PBS solution even up to 15 days (data not 43 l a 44 shown), clearly revealed that the stable π-conjugated backbone of PDA prevents the n 45 46 r leaching out of the encapsulated complex from liposomes for such a long period. Upon u 47 48 encapsulation of RATMP(C) into liposomes, the characteristic MLCT absorption band o 49 J 50 of the complex appeared with a remarkable red-shift of 116 nm (Figure 2a), suggesting 51 w 52 the strong binding interaction between the complex and liposome. This bathochromic e 53 N shift may be ascribed to the partial insertion of the aromatic chromophore of the 54 55 trimethoprim ligand in between the bilayer of liposomes. RATMP(C) complex 56 57 exhibited weak emission in the region of 450 nm to 600 with an excitation wavelength 58 59 of 422 nm in 5% acetonitrile/10 mM PBS solution (pH 7.2). However, the emission 60 17 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 18 of 36 1 2 3 intensity of liposome-bound complex ( , 420 nm;  , 524 nm) is increased due to the ex em 4 5 stabilization of excited state of the complex upon encapsulation (Figure 2b).29 Thus the 6 7 luminescence behaviour of Lip-RATMP(C) supports the partial intercalation of 8 9 complex in between lipid bilayer. t 10 p 11 It was already established that the liposomes with the sizes typically in the range i 12 r of 50-100 nm exhibited weak interaction with plasma proteins, long-term survival in c 13 14 s blood. However, liposomal nanoparticles with the size up to 400 nm have demonstrated 15 u 16 extravasation and accumulation in tumors.47 The well-dispersed liposomal n 17 a 18 nanoparticles with an average size of 167.17 ± 1.76 nm are shown in figure 2c. The M 19 20 spherical morphology of Lip-RATMP(C) was clearly observed by TEM and SEM d 21 22 measurements (Figures 2c and 2d). Moreover, the Energy-Dispersive X-ray (EDX) e 23 t 24 analysis clearly shows the presence of Ru in Lip-RATMP(C) (Figures 2e & 2f). DLS p 25 e 26 measurement indicates the hydrodynamic diameter of Lip-RATMP(C) is 300.8 ± 24.8 c 27 c nm (Figure 2g). Also, the size of Lip-RATMP(C) (300.8 ± 24.8 nm) is higher than that 28 A 29 of bare liposomes without encapsulation (80.1 ± 4.4 nm), revealed the entrapment of 30 y 31 complex into liposomes. The experimentally observed surface charge of Lip- r 32 t 33 RATMP(C) from zeta potential analysis is – 0.4 mV (Table S1), illustrating the stable s 34 i m 35 formulation of liposomes due to electrostatic repulsion between the vesicles. 36 e 37 Furthermore, no significant difference is observed in the zeta potential value of bare h 38 C 39 liposomes (- 0.1 mV), when compared to RATMP(C)-loaded liposomes. All the above 40 results clearly infer the successful encapsulation of RATMP(C) complex into liposomes f 41 o 42 and the obtained size and morphology of Lip-RATMP(C) is suitable for drug delivery 43 l a 44 applications. n 45 46 r u 47 48 o 49 In vitro release study J 50 51 w The In vitro release profile of RATMP(C) from Lip-RATMP(C) was performed 52 e 53 in a simulated physiological environment over 10 days period in order to understand N 54 55 the drug-release ability of Lip-RATMP(C). It is possible that liposomes could release 56 57 the encapsulated complex through the lipid bilayer by diffusion process.48 The less 58 59 amount of complex released (~13%) at 24 h reveals that the complex is properly 60 18 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A incorporated within liposomes up to 24 h in a physiological medium and the burst release of the complex from liposome has not occurred initially. Shen et al reported that ~50% of the encapsulated [Ru(phen) dppz](ClO ) complex was released from 2 4 2 liposomes that consist of dipalmitoylphosphatidylcholine, cholesterol and polyethylene glycol (peg) at 115 h.29 This result demonstrates that the slow release of complex is achieved due to the presence of pegylated bilayer in liposome. Similarly, Lip- RATMP(C) releases ~50% of the encapsulated complex after 110 h (~ 4.5 days), illustrating that conjugated polymeric backbone of polydiacetylene-phospholipid assembly assists the slow-release of complex. Further the observation of a steady increase in the amount of released RATMP(C) with the increasing time up to 150 h (~ 6 days), which is shown in figure 3 indicates that the nanoaggregate releases the complex slowly for such long duration. The release profile has reached a plateau after 180 h (~ 6 days). After that there was no noticeable increase in the release profile observed; it can conclude or seems like that the release process was completed and had reached equilibrium at this late point. To confirm this hypothesis, we followed the dialysis system for a longer time (up to 250 h or ~10 days), but no significant alteration in the release profile is observed. From the above obtained results, it is ensured that the liposomes, which are comprised of PCDA and DMPC, can act as a suitable carrier for slow-release of Ru-arene complexes. 80 60 40 20 0 0 50 100 150 200 250 19 )%( )C(PMTAR desaeleR Page 19 of 36 New Journal of Chemistry 1 2 3 4 5 6 7 8 9 t 10 p 11 i 12 r c 13 14 s 15 u 16 n 17 a 18 M 19 20 d 21 22 e 23 t p 24 25 e 26 c 27 c 28 A 29 30 y 31 r 32 t 33 s 34 i m 35 36 e 37 h 38 C 39 40 f 41 o 42 43 l a 44 n 45 46 r u 47 48 o 49 J 50 51 w 52 e 53 N 54 55 56 57 58 59 60 Time (hours) .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 20 of 36 1 2 3 4 5 6 7 8 9 t 10 Figure 3. In vitro release of RATMP(C) from Lip-RATMP(C). p 11 i 12 r c 13 14 s 15 Interaction with DNA u 16 n 17 a 18 Ru(II)-arene complexes tend to target different biomolecules for exhibiting their M 19 anticancer activity.49 For example, the cytotoxic activity of Ru-arene-ethylenediamine 20 d 21 complexes has been associated with their DNA binding affinity,50a but RAPTA-C 22 e 23 t showed anti-metastatic, and anti-angiogenic activities due to its preferential binding to p 24 25 histone protein in chromatin.50b DNA is a well-known pharmacological target for most e 26 c 27 of the metal-based anticancer drugs. Hence, the ability of the RATMP(C) to effect DNA c 28 A 29 cleavage has been studied by incubating various concentration of complex with 30 y 31 supercoiled pBR322 plasmid (SC) DNA (40 μM) in the absence of any external agents r 32 t 33 and then carrying out the gel electrophoresis technique (Figure 4a, Table S2). Since s 34 i neither the retardation of the mobility of SC DNA nor the retention of SC DNA in the m 35 36 well was observed, the covalent DNA binding of the complex is ruled out.38 In e 37 h 38 comparison with control, ~43% of the SC DNA with an undetectable nicked circular C 39 40 (NC) DNA is observed at 100 μM concentration of complex whereas the complete f 41 o 42 degradation of DNA with undetectable fragments occurs at 200 μM concentration 43 l a 44 (Lane 3 & 4 in Figure 4a). The DNA cleavage activity of Lip-RATMP(C) n 45 46 nanoformulation has been investigated in comparison with the RATMP(C) complex r u 47 48 under identical conditions by using the same gel electrophoresis technique (Figure 4b, o 49 J Table S3). Even though RATMP(C) complex shows the maximum DNA cleavage 50 51 w activity at 200 μM concentration, the nano-formulation, i.e., the complex-loaded 52 e 53 liposome fails to show any DNA cleavage even at the same concentration. The poor N 54 55 DNA cleavage activity of Lip-RATMP(C) indicates that the bilayer of liposome acts as 56 57 a barrier for complex to interact with DNA. So the complex, which is strongly bound 58 59 within a liposome, needs to be released out to exert DNA cleavage activity. Therefore, 60 20 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 21 of 36 New Journal of Chemistry 1 2 3 due to the durable encapsulation of the Ru-arene complex into the liposome, it is 4 5 possible to precise the interaction of Ru-arene complex with DNA rather than random 6 7 attack. 8 9 t 10 p 11 (a) (b) i 12 NC DNA r c 13 14 s 15 u 16 SC DNA n 17 a 18 M 19 1 2 3 4 1 2 3 4 5 6 7 20 d 21 22 e Figure 4. (a) Cleavage of pBR322 DNA (40 μM) by RATMP(C) in absence of an external 23 t 24 agent in 5 mM TrisHCl/50 mM NaCl buffer at 37 oC. Lane 1 : Control DNA; Lane 2 : DNA + p 25 e RATMP(C) (50 M); Lane 3 : DNA + RATMP(C) (100 M); Lane 4 : DNA + RATMP(C) 26 c (200 M) (b) Comparison of DNA cleavage activity of RATMP(C) with Lip-RATMP(C). Lane 27 c 28 1: Control DNA; Lane 2 : DNA + RATMP(C) (50 M); Lane 3 : DNA + RATMP(C) (100 A 29 M); Lane 4 : DNA + Lip-RATMP(C) (100 M); Lane 5: DNA + RATMP(C) (200 M); Lane 30 y 31 6: DNA + Lip-RATMP(C) (200 M); Lane 7: DNA + trimethoprim (200 M). r 32 t 33 s 34 i m 35 As the complex shows efficient hydrolytic DNA cleavage, its DNA binding 36 e 37 affinity was assessed by UV-Vis absorption spectral titration (Figure 5). Upon h 38 C 39 incremental addition of CT DNA to complex (R = [DNA]/[complex] = 25) in 5 mM 40 f 41 Tris-HCl buffer, the decrease in absorption intensity (hypochromism , ~45%) with o 42 43 no red-shift in intra-ligand π–π* transition and MLCT band positions is observed. l a 44 Further, the intrinsic DNA binding constant (K ) is calculated using the equation of n 45 b 46 r Bard et al51 and the value (4.9  103 M-1) (Figure S8) is much lower than those observed u 47 48 o for strong DNA binding agents like EthBr (K , 4.94 × 105 M-1)52 and [(η6-p- 49 b J 50 cymene)Ru(pmpzdpm)Cl], where pmpzdpm is 5-(2- 51 w 52 pyrimidylpiperazine)phenyldipyrromethene (K = 6.9 105 M-1).53 This is apparently e 53 b N 54 due to weak electrostatic interaction between neutral complex and negatively-charged 55 56 phosphate backbone of DNA. Also, the non-planarity of trimethoprim ligand is likely 57 58 to prevent the intercalative interaction of the complex with base pairs of DNA. 59 60 However, the moderate DNA binding affinity of the complex is obviously because of 21 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A 22 ecnabrosbA 0 0 0 . . . 8 4 0 2 4 0 3 0 0 W a v 3 e 6 0 l e n R g R = = t h 2 ( 0 5 4 n 2 m 0 ) 4 8 0 5 4 0 New Journal of Chemistry Page 22 of 36 1 2 3 the hydrophobic interaction of p-cymene and trimethoprim ligands as well as hydrogen 4 5 bonding interaction of –NH groups of trimethoprim ligand with nucleobases of DNA. 2 6 7 8 9 t 10 p 11 i 12 r c 13 14 s 15 u 16 n 17 a 18 M 19 20 d 21 22 e 23 t p 24 25 e 26 c 27 c 28 A 29 30 y 31 r 32 t 33 s 34 Figure 5. Absorption spectra of RATMP(C) (25  10-3 M) in 5 mM Tris-HCl buffer at pH 7.1, i m 35 in the absence (R = 0) and presence (R = 1 - 25) of increasing amounts of CT DNA. (R 36 =[DNA]/[complex]) e 37 h 38 C 39 40 f 41 o 42 43 l a 44 Anti-proliferative activity n 45 46 r Evaluation of healthy cell viability and In Vitro Cytotoxicity u 47 48 o 49 J Since the assessment of biocompatibility is the preliminary step for developing 50 51 potential anticancer therapeutics, the cytotoxicity of RATMP(C) and Lip-RATMP(C) w 52 e 53 in the healthy human embryonic kidney (HEK-293) cells were evaluated by adopting N 54 55 MTT assay (Figure S9). The reported IC value of well-known anticancer drug cis- 50 56 57 platin in HEK-293 healthy cells at 24 h incubation is 4.72 ± 0.07 μM.54a But RATMP(C) 58 exhibits ~80% cell viability even up to 100 μM concentration, illustrating that 59 60 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 23 of 36 New Journal of Chemistry 1 2 3 RATMP(C) appears to be not much toxic in healthy cells compared to cis-platin. 4 5 However, when the concentration of RATMP(C) is increased to 300 μM, it shows ~60% 6 7 cell viability only. But Lip-RATMP(C) retains nearly 80% of cell viability in HEK-239 8 9 cells up to the 300 μM concentration tested. From the results, it is clear that the t 10 p encapsulation of RATMP(C) into liposomes reduces its toxic effect in healthy cells at 11 i 12 r higher concentrations. Therefore the liposome, which is made up of PCDA and DMPC, c 13 14 s could serve as a biocompatible drug carrier. 15 u 16 n 17 a 18 Table 3. In vitro cytotoxicity studies for RATMP(C) and Lip-RATMP(C) against A549 human M 19 lung adenocarcinoma, MCF-7 human breast carcinoma, HepG2 human liver carcinoma cell 20 lines for 24 h incubation. Inhibitory concentration (IC ) values are in μM. (Data are mean  d 21 50 22 SD of three replicates each) e 23 t 24 aIC , M p 50 25 e 26 c A549 MCF-7 HepG2 27 c lung adenocarcinoma breast carcinoma liver carcinoma 28 A 29 30 y 31 RATMP(C) > 150 > 300 48.8  5.1 r 32 t 33 Lip-RATMP(C) > 150 > 300 35.2  3.7 s 34 i trimethoprim > 300 > 300 > 150 m 35 36 e 37 h 38 aIC = concentration of drug required to inhibit growth of 50% of the cancer cells (in μM) C 39 50 40 f 41 o 42 The In vitro cytotoxic activity of trimethoprim, RATMP(C) and Lip-RATMP(C) 43 l a 44 has also been investigated against three different cancer cell lines, namely, A549 human n 45 46 r lung adenocarcinoma, MCF-7 human breast carcinoma, HepG2 human liver carcinoma u 47 48 cell lines by using MTT assay (Table 3). Trimethoprim is found to be insensitive to all o 49 J 50 these three cell lines. Similarly, both the complex and nanoformulation fail to show 51 w 52 marked cytotoxicity against A549 lung adenocarcinoma (IC : > 150 μM) and MCF-7 50 e 53 N 54 breast carcinoma (IC 50 : > 300 μM). In contrast, both RATMP(C) (IC 50 : 48.8(5.1) μM) 55 and Lip-RATMP(C) (IC : 35.2(3.7) μM) show efficient cytotoxicity against human 56 50 57 liver carcinoma (HepG2) cells. However, the cytotoxicity of compounds towards 58 59 60 23 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 24 of 36 1 2 3 HepG2 liver cancer cells is much lower than that observed in cisplatin (IC : 0.05 50 4 5 μM).54b 6 7 8 Interestingly, Lip-RATMP(C) shows a higher cytotoxicity than the RATMP(C) 9 complex against liver cancer cells, revealing that encapsulation of complex into t 10 p 11 liposomes enhances its cytotoxicity. It appears that the complex-loaded liposome, i 12 r c 13 because of its higher hydrophobic interaction through the phospholipid bilayer, would 14 s 15 pass of the cell membrane more easily than the non-encapsulated complex.55 Further, u 16 n 17 on account of biodegradability of liposome in cellular environment,56 the complex- a 18 M 19 loaded liposome would release the intact form of complex in a slow manner (cf. above) 20 21 at various organelles inside the cell. DNA would be a probable target for the released d 22 e 23 complex to exhibit its cell killing activity, as it cleaves DNA even in absence of any t p 24 external agent. The cytotoxicity of complex may be traced to the suitable hydrophobic 25 e 26 c and hydrogen bonding potency ligands incorporated as a DNA recognition elements in 27 c 28 the complex. Nevertheless, like RAPTA(C) complex, the anticancer activity of released A 29 30 complex as a result of its preferential binding to specific cancer inhibiting protein or y 31 r 32 enzyme cannot be excluded. Further investigation is required to confirm the mechanism t 33 s 34 of cytotoxicity and to detect the selectivity of compounds towards liver cancer cells i m 35 36 rather than breast and lung cancer cells. e 37 h 38 C 39 40 f 41 o 42 43 l a 44 Evaluation of apoptosis using AO/EB and PI staining assay n 45 46 The evaluation of characteristic morphological changes occurred in HepG2 r u 47 48 human liver carcinoma cells after treating them with IC 50 concentration of o 49 J RATMP(C) and Lip-RATMP(C) provides information about the molecular 50 51 w mechanism of cell death.57 For this purpose, Acridine Orange (AO)/Ethidium 52 e 53 bromide (EB) staining assay was used and the fluorescent microscopic images N 54 55 are shown in Figure 6. Acridine orange (AO) permeates the cells and makes the 56 57 nuclei appear green. Ethidium bromide (EB) is taken up by cells only when 58 59 cytoplasmic membrane integrity is lost and stains the nucleus red. Depending 60 24 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 25 of 36 New Journal of Chemistry 1 2 3 upon the fluorescence emission and morphological features of chromatin 4 5 condensation in the AO/EB stained nuclei, the cytological changes observed are 6 7 generally classified into four types: (i) viable cells that have uniformly green 8 9 fluorescing nuclei with highly organized structure; (ii) early apoptotic cells that t 10 p have green fluorescing nuclei but where perinuclear chromatin condensation is 11 i 12 r visible as bright green patches or fragments; (iii) late apoptotic cells that have c 13 14 s orange to red fluorescing nuclei with condensed or fragmented chromatin; and 15 u 16 (iv) necrotic cells, swollen to large sizes, that have uniformly orange to red n 17 a 18 fluorescing nuclei with no indication of chromatin fragmentation.18 The results M 19 20 obtained from AO/EB staining assay indicate that the complex and d 21 22 nanoformulation induce both apoptotic and necrotic mode of cell death, but e 23 t 24 mainly through apoptosis. Further, the apoptosis-inducing ability of compounds p 25 e has been evaluated by adopting fluorescence microscopic analysis of PI-stained 26 c 27 c HepG2 cancer cells. Morphological changes such as cell shrinkage, chromatin 28 A 29 condensation, membrane blebbing, DNA fragmentation, and formation of small 30 y 31 apoptotic bodies observed using PI staining assay also confirm the apoptotic r 32 t 33 mode of cell death. The apoptosis-inducing ability of nanoformulation and s 34 i m 35 complex follow the trend Lip-RATMP(C) (68%) > RATMP(C) (52%) (Figure 36 e 37 S10), which is consistent with their cytotoxic activity against HepG2 liver h 38 carcinoma cell lines. Similar observations have been made by Shen et al29 earlier C 39 40 for Liposomal-Ru (Ru is [Ru(phen) (dppz)](ClO ) ), which induces 75.9% f 41 2 4 2 o 42 apoptosis in MDA-MB-231 breast cancer cells, while less than 5% displayed 43 l a 44 signs of apoptosis in Ru-treated cells. The apoptosis-inducing ability of n 45 46 liposomal-Ru(arene) complex is higher than that of non-encapsulated complex r u 47 48 confirming that the cellular-internalisation potency of the complex is enhanced o 49 J 50 after its encapsulation into liposomes. Also, on account of the enhanced 51 w 52 permeability and retention effect of nanoformulation, it induces a higher extent e 53 of apoptosis than free complex in liver cancer cells. N 54 55 56 57 58 59 60 25 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 26 of 36 1 2 3 4 5 6 7 8 9 t 10 p 11 i 12 r c 13 14 s 15 u 16 n 17 a 18 M 19 20 d 21 22 e 23 t p 24 25 Figure 6. Morphological evidences of apoptosis by AO/EB dual staining and PI Staining assay. e 26 Fluorescent microscopic images of human liver carcinoma (HepG2) lines after treatment with c 27 c IC concentration of RATMP(C) and Lip-RATMP(C). 50 28 A 29 30 y 31 r 32 Antibacterial activity t 33 s 34 i m 35 Since most of the cancer patients are easily affected by bacterial infection, it is 36 e 37 an ideal approach to assess the antimicrobial activity of anticancer drug candidates h 38 instead of designing two different drugs for cancer treatment and bacterial infection C 39 40 individually. The antimicrobial activity of RATMP(C) and Lip-RATMP(C) was tested f 41 o 42 on representatives of pathogenic microorganisms such as Gram-positive bacteria S. 43 l a 44 aureus and Gram-negative bacteria P. aeruginosa. These two strains were selected n 45 46 because they have identified as two of the most common pathogens for causing r u 47 48 nosocomial infections in patients with cancer.58 Also, these strains showed In vitro o 49 J 50 susceptibility to trimethoprim alone or combination with sulphonamides.59 The results 51 w 52 of bactericidal activity of trimethoprim, RATMP(C) and Lip-RATMP(C) are shown in e 53 figure S11. The antimicrobial stroke of RATMP(C) against P. aeruginosa is identified N 54 55 to be high at 50 µM. Also, the bactericidal activity of complex against S. aureus was 56 57 gradually increased with the increase of concentration of complex in a dose-dependent 58 59 manner (Table 4). RATMP(C) exhibits antibacterial activity with similar potency as 60 26 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 27 of 36 New Journal of Chemistry 1 2 3 trimethoprim in both bacterial strains, illustrating that the coordination of trimethoprim 4 5 to Ru(II)-p-cymene moiety does not alter its antimicrobial activity significantly. Like 6 7 trimethoprim, the mechanism of antibacterial activity of RATMP(C) may be ascribed 8 9 to inhibition of bacterial DHFR as the complex contains pteridine structural analogue t 10 p of dihydrofolic acid.60 On the other hand, Lip-RATMP(C) is found to be insensitive 11 i 12 r even in the concentration of 300 μM tested (data not shown). This indicated that the c 13 14 s PCDA/DMPC assembly is not able to fuse with outer membrane of bacteria and fails to 15 u 16 permeate into cellular membrane to exert antibacterial activity. Thus, different n 17 a 18 strategies like receptor-mediated delivery for targeting bacteria might be necessary for M 19 20 Lip-RATMP(C) to obtain improved antibacterial activity. d 21 22 e 23 t 24 p 25 e 26 c 27 Table 4. Antibacterial activity of test agents against Gram-positive S. aureus and Gram- c 28 A negative P. aeruginosa for 24 h incubation at 37 °C using agar disk diffusion method. 29 30 y 31 r 32 Bacterial strains used Tested Average diameter of zone of inhibition t 33 s compound (mm) including disk (6 mm) 34 i m 35 36 e 37 25 µM 50 µM 75 µM 100 µM h 38 C 39 40 f 41 o trimethoprim 20±0.1 23±0.2 22±0.32 29±0.1 42 43 2 3 5 l P. aeruginosa a 44 n 45 RATMP(C) 22±0.3 24±0.5 21±0.42 20±0.2 46 r 47 5 0 2 u 48 o 49 J trimethoprim 21±0.6 23±0.8 24±0.11 26±0.3 50 51 4 8 7 w 52 S. aureus e 53 RATMP(C) 19±0.2 20±0.1 20.5±0.1 23±0.1 N 54 55 5 5 4 0 56 57 58 59 60 27 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A New Journal of Chemistry Page 28 of 36 1 2 3 Conclusions 4 5 One of the aims of this present study is to design the dual functionality organometallic 6 7 Ru-arene complex that exerts anticancer and antimicrobial properties. For this purpose, 8 9 we synthesized the half-sandwich ruthenium complex of the type [Ru(η6-p- t 10 p cymene)(trimethoprim)Cl ] by incorporating the antibacterial drug trimethoprim to the 11 2 i 12 r (η6-p-cymene)Ru fragment that has been well-reported for its efficient anticancer c 13 14 s activity. The coordination geometry around Ru(II) metal centre is described as a typical 15 u 16 “piano-stool” pseudo-octahedral. The second aim of this work is to encapsulate the n 17 a 18 isolated complex into the liposome to further enhance its pharmacological properties. M 19 20 Liposomal-Ru nanoaggregate was prepared by using self-assembly of an appropriate d 21 22 concentration of 2-ditetradecanoyl-sn-glycero-3-phosphocholine and 10,12- e 23 t 24 pentacosadiynoic acid in presence of complex isolated. The π-conjugated backbone of p 25 e polydiacetylene facilitates the slow-release of complex from liposome. Hence, only 26 c 27 c 13% and 50% of encapsulated complex are released from liposome over 24 and 110 h, 28 A 29 respectively under physiological conditions. Prior to assess the biological activity, the 30 y 31 interaction of complex and nanoaggregate with DNA has been carried out since DNA r 32 t 33 is considered as an important pharmacological target for metal-based drugs. The s 34 i m 35 nanoaggregate exhibits less toxicity in normal kidney cells, illustrating that 36 e 37 PCDA/DMPC liposomes could act as a biocompatible drug delivery system. The h 38 complex and nanoaggregate are remarkable in displaying more cytotoxic activity C 39 40 against liver cancer cells rather than breast and lung cancer cells. Notably, the f 41 o 42 nanoaggregate demonstrates more cytotoxic activity and apoptosis-inducing activity in 43 l a 44 liver cancer cells in comparison with that of complex alone. Thus, the development of n 45 46 such a liposome-encapsulated Ru(arene) complex containing the bioactive ligand could r u 47 48 potentially alleviate systemic toxicity of the complex while maximizing the anticancer o 49 J 50 activity. Nevertheless, the nanoaggregate was found to be ineffective to enhance the 51 w 52 antibacterial activity of the complex, which exhibited potent and dose-dependent e 53 antibacterial activity. N 54 55 56 Supplementary material 57 58 59 60 28 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A Page 29 of 36 New Journal of Chemistry 1 2 3 CCDC 2004416 contains the supplementary crystallographic data for this paper. 4 5 The data can be obtained free of charge from the Cambridge Crystallographic Data 6 7 Centre via https://www.ccdc.cam.ac.uk. 8 9 Conflicts of interest t 10 p 11 i 12 There are no conflicts of interest to declare. r c 13 14 Acknowledgments s 15 u 16 n 17 M.G sincerely thanks Science and Engineering Research Board (SERB), New Delhi for a 18 M a Start-up research grant (YSS/2015/000403/CS) and Junior Research Fellowship to 19 20 D.G. We gratefully acknowledge SRM Institute of Science & Technology, d 21 22 e Kattankulathur for providing infrastructure and instrumental facility. CS is thankful to 23 t p 24 SRM Institute of Science & Technology, Kattankulathur for providing research 25 e 26 fellowship. We acknowledge DST-FIST (fund for the improvement of S&T c 27 c 28 infrastructure) for financial assistance for Department of Chemistry, SRM Institute of A 29 30 Science & Technology, Kattankulathur (No.SR/FST/CST-266/2015(c)). Authorship y 31 r 32 agreed by all authors except Dr Suvankar Ghorai who could not be contacted. t 33 s 34 i References m 35 36 e 37 1. References h 38 C 39 1. a) E. R. Jamieson and S. J. Lippard, Chem. Rev., 1999, 99, 2467–2498. b) S. 40 41 Dilruba and G.V. Kalyda, Cancer Chemother.Pharmacol., 2016, 77, 1103–1124. o f 42 43 l 2. L. Kelland, Nat. Rev. Cancer, 2007, 7, 573−584 a 44 n 45 46 3. M. L. Rothenberg, D. R. Carbone and D. H. Johnson, Nat. Rev. Cancer, r u 47 2003, 3, 303−309. 48 o 49 J 4. G. Housman, S. Byler, S. Heerboth, K. Lapinska, M. Longacre, N. Snyder 50 51 and S. Sarkar, Cancers, 2014, 6, 1769−1792. w 52 e 53 5. a) T. Fuereder and W. Berger, ESMO Open, 2017, 2, e000239. b) H. A. N 54 Burris, S. Bakewell, J. C. Bendell, J. R. Infante, S. F. Jones, D. R. Spigel, G. 55 J. Weiss, R. K. Ramanathan, A. Ogden and D. D. Von Hoff, ESMO Open, 56 57 2016, 1, e000154. 58 59 60 29 .MA 41:24:01 0202/12/01 no thcertU tietisrevinU yb dedaolnwoD .0202 rebotcO 02 no dehsilbuP View Article Online DOI: 10.1039/D0NJ03664A