Practical pilot tests have been conducted to remove heavy metals, which gave highly satisfactory results. Challenges such as agglomeration, sedimentation, magnetic susceptibility, sorption to other fine materials in aqueous solution and toxicity of microbiomes have been reported. Emerging studies have highlighted the prospects of industrial level application of nano zero valent particles for the remediation of heavy metals and other pollutants from various industries.For the past 15 years, nanoscale metallic iron (nZVI) has been investigated as a new tool for the treatment of heavy metal contaminated water. The removal mechanisms depend on the type of heavy metals and their thermodynamic properties. A metal whose redox potential is more negative or close to the reduction potential of Fe(0) is removed by the reduction process, while the others will be mediated by precipitation, complexation or other sorption processes. This review summarises our contemporary knowledge of nZVI aqueous chemistry, synthesis methods, mechanisms and actions  of heavy metal  removal and challenges of nZVI practical applications. Its inner core (iron(0)) has reducing ability towards pollutants, while the iron oxide (FeO) outer shell provides reaction sites for chemisorption and electrostatic interactions with heavy metals. Emerging studies highlighted that nZVI surfaces will have negatively charged species at higher pH and have good affinity for the removal of positively charged species such as heavy metals. Different sizes, shapes and properties of nZVI have been produced using various methods. Ferric salt reduction methods are the most common methods to produce stable and fine graded nZVI. Higher uptake of copper removal mechanisms of nZVI from aqueous solution.Heavy metals (Cd Many technologies have been developed to reduce these pollutants before reaching to water bodies. Nanotechnology is among the recently introduced heavy metal removal techniques that has attracted a lot of attention of many scientists.1,14,15 Nano-zero valent iron (nZVI) has Fe(0) core and Fe oxide outer shell, wherein the core has reducing ability and the outer shell acts as a reaction site for chemisorption and electrostatic interactions. Mechanisms such as reduction, absorption, precipitation and mineralization have played a role in the removal of heavy metals from aqueous phases using nZVI.1,15 When dissolved in water nZVI exhibits ligand like properties. At low pH, iron oxide shell is positively charged and attracts anions like phosphates and sulphates, and at high pH it becomes negatively charged and attracts cations like metal ions. Heavy metals are sequestered onto the solid nZVI surface after the reaction.1 In this paper, different articles that have been published on various methods of nZVI synthesis, a laboratory scale and field level practical tests of heavy metal removal capacity of nZVI were collected and reviewed.Nano zero valent iron was extensively studied for its ability to remediate heavy metal contaminated wastewater effluents from different industries. It's less toxicity and comparative abundance in nature led nZVI to wide applications in the field of environmental pollution abatement.2.2.116,17 It has been reported in the literature that iron is the second most abundant metal next to aluminium in igneous rocks. The element is a principal constituent of many igneous rocks, especially those containing basic silicate minerals. Divalent iron links the chains of silicon-oxygen tetrahedra in minerals such as pyroxenes and amphiboles and links individual tetrahedron in the structure of fayalite. Trivalent iron replaces aluminium in some silicate minerals. Also, iron is common in the form of oxide and sulphide.Iron is relatively abundant in the earth's crust.Sedimentary rocks contain iron in various forms, but ferric oxides are perhaps more common. Geochemical data show that iron weathered out of solid minerals is not retained for long in solution in water but is redeposited as solid oxides or hydroxides. Iron occurs in two oxidation states, the divalent or ferrous form and the trivalent or ferric form.Iron in aqueous solution is subject to hydrolysis. Iron hydroxides are formed, especially the ferric form with very low solubility. The retention of iron in solution is consequently affected by the pH of the solution. In most natural waters, the pH is not low enough to prevent hydroxides from forming, and under oxidizing conditions, practically all the iron is precipitated as ferric hydroxide. Another important feature of the chemical behaviour of iron in solution is its tendency to form complex ions with inorganic ligands .e.g., metal ions). At higher pH above its iso-electric point, zero-valent iron can serve as very effective electron donors  with nZVI (Fe2+/Fe) standard reduction potential (E0) of −0.44 V, which is lower than many metals such as Pb, Cd, Ni, and Cr. As a result, it will then bind heavy metals.In natural water, ZVI nanoparticles can exhibit metal-like or ligand-like coordination properties depending on the solution chemistry. Iron(0) particles are covered with iron oxides. At lower tract when the pH is lower than its point of zero charge pH (<pHzpc = 8), the iron oxides (nZVI surface cover) are positively charged whereas when the solution pH is above the iso-electric point, the oxide surface becomes negatively charged and can form surface complexes with cations (2.20), also referred to as zero-valent iron, is well recognised as being highly susceptible to oxidation in aqueous media. Its oxidation is considered to occur primarily through an electrochemical process, with anodic and cathodic components. The anodic reaction involves the dissolution of Fe (forming soluble ionic products or insoluble oxide/hydroxide) and is coupled with the reduction of redox amenable species at the cathode. In natural waters, the primary components available for oxidation reactions are dissolved oxygen (DO) and water, with the former being thermodynamically favoured 2+) is a primary product from these reactions and, in turn, can undergo further oxidative transformation (Ferrous ion (Feormation .32Fe(s)22 g−1) rapidly facilitates the chemically reducing conditions when the mass of material is added to an aqueous system  or partially oxidized magnetite (Fe3O4). Nanoparticles formed via nucleation of metallic vapor also contain γ-Fe2O3 and Fe3O4, with more γ-Fe2O3 for smaller particles due to the higher surface-to-volume ratio and rapid surface oxidation. On the other hand, particles produced by hydrogen reduction of goethite and hematite particles reportedly have only Fe3O4 in the shell.21,22 The presence of wustite (FeO) has also been noted. It is not clear from the existing literature whether variations in the shell structure and composition have any effect on the iron nanoparticle reactivity, aggregation, and transport.In actual conditions, nZVI is covered with an oxide layer . Accordi3.24 The choice of synthesis method influences both the size and shape of the nano particles produced.There exist several physical and chemical methods for the synthesis of nZVI such as grinding, abrasion, lithography, nucleation from homogeneous solutions or gas, annealing at elevated temperatures and reacting with reducing agents. These synthesis methods are grouped under two broad approaches: the bottom-up and top-down approaches. The bottom-up approach involves assembling individual atoms and molecules to form nano-sized structures; it uses a wide range of reductants to convert dissolved iron in solution to nZVI. Whereas, the top-down approach on the contrary involves the crushing of bulk particles  of iron to fine nano-sized particles by mechanical or chemical ways.3.14). The borohydride solution prepared from concentrated ethanol and NaBH4(s) is slowly added into the iron (ferric or ferrous) salt solution while stirring vigorously under anaerobic conditions (25 As shown in iii) reduced to iron(ii); then it converts to nZVI in the presence of excess reducing agents.The synthesis of nZVI can be achieved by reacting iron salts or compounds with a strong reducing agent such as sodium borohydride . The particles that are produced in these methods have an average particle size ranging from 50 to 300 nm with a specific area of about 7.55 m2 g−1.27 The reaction proceeds according to This process uses gases such as H3.32, H2, CO, and Ar.28,29One synthesis method which is well-known for producing high quality particles with more narrow size distribution is the thermal decomposition of an iron precursor. This method is quite similar to the gas phase reduction previously explained. The iron precursor  is reduced at high temperatures (>500 °C) in the presence of gaseous reducing agents. The reducing agents used for this synthesis include N3.4et al. (2016),29 the ultrasound assisted nZVI syntehsis method was first developed and tested by Tao et al. in the State Key Laboratory for RSA, Institute of Metal Research, Chinese Academy of Sciences in 1999. The method helps to enhance the physical and chemical properties of nZVI  and the shell, i.e., the oxide layer of Fe(ii) and Fe(iii), which is formed due to the rapid metallic iron oxidation in the environment  acts as an electron donor with the following anodic reaction: Fe → Fe2+ + 2e−.1,37 The outer layer of iron (hydro)oxide acts as an effective adsorbent of pollutants, including heavy metals.20,38 In water, Fe(0) nanoparticles can show the coordination properties of a metal or ligand depending on the pH of the solution. At pH > 8, the surface of nanoparticles becomes negatively charged and can form surface complexes with heavy metal cations.The availability of active sites and/or many functional groups on nZVI surface and sub surface has been confirmed.ironment . Metalli39 Heavy metals such as nickel (Ni2+) and lead (Pb2+) were removed from aqueous solutions by reducing to Ni0 and Pb0 and adsorbing these cations on iron (hydr)oxide shell.1 Also, Pb2+ was precipitated to Pb(OH)2 and separated from the solution in the form of precipitant. But Pb(OH)2 was changed to hydroxide complex at higher pH and setting of the appropriate optimal pH for the lead hydroxide precipitant formaiton was required.40 On the other hand, copper adsorption study on nZVI showed the removal of Cu2+ from solution by chemical reduction to an elemental form and further sorption on the oxidized surface of nZVI.41,42 In general, other competing heavy metal ions affect the reduction process and the co-existing ions such as phosphates, bicarbonates/carbonates, sulphates and nitrates found in solution affect (co)precipitation.43,44The reduction of heavy metals involves two distinct mechanisms; first, the heavy metal is reduced when it comes in contact with Fe(0) in an aqueous solution. Then, the heavy metal is adsorbed onto the core–shell structure of nZVI followed by its gradual reduction by Fe(0).45,46The main adsorption mechanisms of common heavy metals found in the surface, ground and wastewater depend on their aquatic chemistry. However, the removal mechanism is mainly dominated by adsorption and other processes such as reduction, hydroxide precipitation and electron transfer. Studies showed that the heavy metals in the solution might undergo one or more of the processes such as surface complexation, physisorption, precipitation and/or reduction.E0 = −0.44 V) undergo reduction; nZVI serves as an effective electron donor for the metallic ions to be reduced.47 The metals whose redox potential (E0) is more negative or close to that of Fe0 (such as Cd) are removed by sorption and/or precipitation.20The heavy metals possessing lesser standard redox potential than nZVI  of Cd, Cu and Pb ions was 4.33–5.56, 5.40–6.94 and 5.41–6.95 mg g−1, respectively. The number of adsorbents (nZVI) to be added affects the rate of heavy metal removal. In another study reported by Bagbi et al., almost 100% lead metal ion removal has been recorded using nZVI at following experimental conditions: lead concentration: 50 mg L−1; equilibrium time: 15 min; pH 5–6; temperature: 25 °C and NZVI dose: 0.4 g L−1.49ZVI nanoparticles have been tested for removal of various heavy metals. It was reported that copper, cadmium and lead were efficiently removed by nZVI adsorbents.50 In the report, it was justified that increasing contact time, pH and nZVI dose increases the removal of the contaminant. However, adsorbate concertation has an inverse relation with the removal capacity. Ahmed et al. (2017)50 have reported about 98% of lead removal from water after 10 hours of contact time. The authors reported that the physical properties, magnetic susceptibility, and conductance of nZVI were correlated with adsorption efficiency.In this study, it was justified that increasing the pH promotes the rate of adsorption and lead removal. Lead metal ion removal experiment from wastewater has indicated that the pH of the solution where lead ion is found affects the state or species of nZVI adsorbent used for adsorption; at lower pH, the surface oxide layer is positively charged where it is dominated by protonated ions and attract only the positive ions, whereas at higher pH its surface becomes negatively charged and forms surface complexes with heavy metals such as Cd, Cu, Pb and others.5.2via an oxidation–reduction reaction in which Fe2+ behaves as a reducing agent. The standard reduction potential of Cu  is well above that of Fe , and consequently, the uptake of Cu2+ ions would be expected to primarily take place via a redox mechanism. The study result reported by Li and Zhang confirmed that Cu2+ ions are reduced to Cu0 upon exposure to nZVI. The authors haves cross confirmed the actions by analysing the distribution of Cu on the surface of iron nanoparticles  removal efficiency of nZVI at operating conditions of 10.20 g L−1 nZVI and an agitation time of 100 min was greater than 96%, and achieved an uptake capacity of 250 mg g−1.45 Li et al. (2017)67 reported a 99% copper removal efficiency from aqueous solution in the presence of other heavy metals. This removal efficiency is very high as compared to the removal capacity of Cu(ii) using other adsorbents such as ZVI/sand mixture, Fe2O3 nanoparticles, grafted silica, wheat bran and spirogyra species  ion under HRT of 6.8 to 34 h, and a volumetric loading rate of 10 g Cu per m3 per day. The author has achieved a removal capacity of 0.029 g Cu per g Fe.51Komnitsas  reactor . Accordi5.32+) removal.38Study has reported Cd metal ion removal capacity of nano zero valent iron (nZVI) by comparing with the removal capacity of other adsorbents that were investigated by other authors . Accordi−1, pH of 6.92, adsorbent dose of 0.56 g L−1 and contact time of 40.42 min. The nZVI formed on the biochar via adsorption and complexation processes increased the ability of Cd removal than the newly produced biochar–nZVI composite adsorbent.55 It was also showed that the equilibrium cadmium adsorption capacity of zero valent iron nano particles with 0.5 g L−1 reaches up to 213 mg g−1 at 285 K and 225 mg g−1 at 333 K.35In 2018, Mahboub Saffari reported a comparative study of cadmium ion adsorption of nZVI to walnut shell biochar modified nZVI; the maximum predicted Cd removal efficiency of the nZVI supported biochar was about 99.72% at initial Cd metal ion concentration of 70.78 mg L5.441 increasing surface area to volume ratios,62 and reducing the size of particles.63 Various researches have been conducted; most of the studies relied on the formation of nZVI supported composites.The adsorption performance of nZVI has been improved by increasing the anionic surfactants for cationic pollutant removal,64 nZVI has small particle size and high surface energy, which can adsorb heavy metals or cationic pollutants easily, but it is very difficult to separate it from either sludge or effluent.65 Coating Pd on nZVI surfaces has improved the interstitial characteristics of nZVI;66 the co-existing organic and inorganic substances impact each of the modified/immobilized nZVI–composites via various mechanisms and reduce the targetted heavy metals removal action from the solution. Hence, modifying the nZVI via additional support material to improve the effect of co-existing interfering substances is helpful.The addition of partially acid activated sepiolite on nZVI improved the dispersibility of the adsorbent materials and also formed a porous oxide that can further enhance the heavy metal entrapping action.6.6.1et al. (2014)45 conducted a pilot test for the removal of copper using nZVI (−1 Cu(ii) with a total of 55 kg of nZVI. A completely mixed reactor of 1600 L was operated continuously with flow rates ranging from 1000 to 2500 L h−1. The average Cu(ii) removal efficiency was greater than 96% with 0.20 g L−1 nZVI and a hydraulic retention time of 100 min.45Li ing nZVI . In thei3 per day for Cu(ii) removal, surpassing the loading rates of conventional technologies by more than one order of magnitude. The average removal capacity of nZVI for Cu(ii) was 0.343 g Cu per gram of Fe. The Cu(ii) removal efficiency can be reliably regulated by the solution Eh, which in turn is a function of nZVI input and hydraulic retention time. The ease of separation and recycling of nZVI contribute to process up-scalability and cost effectiveness. As described in ii) was reduced to metallic copper and cuprite (Cu2O).The nZVI reactor achieved a remarkably high volumetric loading rate of 1876 g Cu per m6.2et al. (2016)44 to evaluate a multi-stage system consisting of lime (ii) and Zn(ii) in wastewater generated by a smelting plant in China. The wastewater contained highly hazardous metal ions ). The treatment system was constructed on-site, and the study was conducted for a period of 6 days. Wang et al. (2016) designed pilot test reactors to compare the removal of lead and zinc using nZVI line separately (Another field study was conducted by Wang  of lime  and nZVIparately .2), the most widely used heavy metal precipitant, for Pb(ii) and Zn(ii) removal field experiments (−1) of Pb(ii) could not be achieved via lime precipitation alone  were easily attained using nZVI due to its multifunctional properties and good tolerance for influent fluctuation enabled by its inherent pH-stabilizing nature  was compared with lime (Ca(OH)eriments . The teson alone  due to ton alone  caused bg nature .et al. (2016), the concentrations of the heavy metals (lead and copper) were much lower than those predicted by the theoretical solubility of metal hydroxides; hydroxide precipitation cannot solely account for the efficient removal, and the combined effect of sorption presorption  and chemsorption  in the s6.3et al. conducted a pilot test to evaluate nZVI efficiency for various heavy metal removal at the nZVI technology-based treatment facility, located in Jiangxi, China and operated by the Jiangxi Copper Company, which showed a promising result for copper ion removal from copper containing wastewater.69 The nZVI treatment plant was constructed in April, 2012, and served as a pre-treatment to remove arsenic and other metals such as copper(ii) from the wastewater . Wastewater samples were collected daily at the outlet of each treatment unit in the process, such as the outlets of equalization basin and nZVI reactor. Solid samples (spent nZVI) were collected from the nZVI reactor. According to the pilot test, quick removal of the four target ions was observed after nZVI addition; 99% of copper, 90% of arsenic, zinc and nickel were removed in less than 10 min. The removal efficiencies are all greater than 96% after 60 min. Increasing nZVI dosages improves the effluent quality and the concentrations of the target ions after filtration meet the local discharge guidelines. The fast and simultaneous removal of different heavy metals using nZVI is the major reason that nZVI was selected and approved for field application.Li stewater . The tre7.70 Nanoparticles adsorbed on cell membranes can block cellular ducts, cause structural changes to the membranes, or inhibit mobility and nutrient intake and result in death of the bacteria.Studies showed that nZVI can be adsorbed on cell membranes of bacteria, or penetrate through them, which often leads to disturbances in the functioning of the cell.2+ to nZVI toxicity is higher under anaerobic conditions than under aerobic conditions.71 Research suggested both modifications and limiting the direct contact of the nZVI surface with cells to reduce the toxicity.Another often-suggested mechanism contributing to nZVI toxicity involves the generation of highly reactive oxygen species, which accumulate in the cell environment and denature macromolecules including lipids, proteins, and nucleic acids, damaging intracellular structures and eventually leading to cell death. Because under aerated conditions, Fe oxidizes more rapidly than it would under anaerobic conditions, the contribution of Fe14 Some of the other major challenges identified with the use of nZVI are the rapid aggregation of the particles, passivation (quick oxidation by non-target compounds), sorption to other materials and rapid sedimentation (because of its high density/7800 kg m−3) that consequently limits the mobility of nanoparticles in the aquatic environment.15,25The application of nZVI works best under anaerobic conditions since it is otherwise quickly transformed to iron oxides under aerobic conditions. Any modification to the surface such as oxidation would therefore affect the performance of the particles.3+ to Fe2+ and gives an opportunity for the targetted pollutants to be oxidized.72Pure nZVI agglomerate easily. It is also very sensitive towards oxidation and reduces the electron donation during remediation of heavy metal containing wastewater. nZVI can be prepared in many ways, however, it is easily oxidized and difficult to preserve. Many of the preparation methods take place under strict conditions, and hence, it is very difficult to proceed to large scale production. However, in general, it is a very promising reductant for cationic pollutants such as metal ions and cationic dyes, which can also be used in other heavy metal remediation. The heavy metal removal mechanisms are limited not only to reduction and adsorption but also to oxidation. The Fenton reaction taking place in the aqueous solution change Fe8.For the treatment of contaminated water, growing theoretical and empirical evidence has proven nZVI as both highly effective and versatile. The sorption, complexation and reduction processes played a great role for the removal of heavy metals. In recent years, there have been significant innovations in terms of manufacture techniques, physicochemical functionalisation and enhancements in sub-surface stability of nZVI. The tests that have been done on the removal activity of Pb, Cd and Cu showed the possibility of applying ZVI nanoparticles at field scale. However, some of its properties such as susceptibility to oxidation and formation of iron oxide, aggregation and sediment-ability need to be studied.Various pilot scale tests for the removal of heavy metals have been conducted. All the tests followed the concept of green chemistry to treat wastewater and recover heavy metals. Practically, the pilot test was operated in continuous mode. To make the process affordable, nZVI was recirculated and reused. The nZVI process presented is designed for large-scale applications and is easy-to-operate. The results of the reviewed practical pilot test suggested that this nZVI process is much more efficient than conventional technologies of wastewater treatment, and some economic minerals such as copper could be easily recovered from the wastewater using this process. In general, the technology has showed satisfactory and promising results for large scale industrial wastewater treatment.Mekonnen Maschal Tarekegn has contributed in compiling articles/literatures, reviewing and manuscript preparation.Andualem Mekonnen Hiruy and Ahmed Hussen Dekebo have contributed in reviewing and correcting the manuscript.We declare that there are no conflicts related to this article.


We identified that OVCD cells are under oxidative stress due to high hydrogen peroxide (H2O2) levels and low glutathione peroxidase and thioredoxin 1. Furthermore, OVCD cells have increased glycolysis activity and mitochondrial respiration when compared to immortalized ovarian cells (hTER7) and parental cancer cells (OV90). Our goal was to study how ovarian cell growth depends upon the redox state of the cell; hence, we used MnP (BMX-001), a redox-active MnSOD mimetic, as a molecular tool to alter ovarian cancer redox state. Interestingly, OVCD cells preferentially uptake MnP relative to OV90 cells which led to increased inhibition of cell growth, glycolytic activity, OXPHOS, and ATP, in OVCD cells. These effects were further increased when MnP was combined with carboplatin. The effects were discussed with regard to the elevation in H2O2 levels, increased oxidative stress, and reduced Nrf2 levels and its downstream targets when cells were exposed to either MnP or MnP/carboplatin. It is significant to emphasize that MnP protects normal ovarian cell line, hTER7, against carboplatin toxicity. Our data demonstrate that the addition of MnP-based redox-active drugs may be used (via increasing excessively the oxidative stress of serous ovarian cancer cells) to improve cancer patients' chemotherapy outcomes, which develop resistance to platinum-based drugs.We have employed a redox-active MnP (MnTnBuOE-2-PyP The level of prooxidants such as ROS (reactive oxygen species) and RNS (reactive nitrogen species), antioxidant proteins, redox thiol couples, repairment proteins, and metal ions will alter the balance of redox state. This redox imbalance in favor of prooxidants leads to persistent oxidative stress. Several investigators have proposed redox imbalance toward oxidative stress as a mechanism for several cancer features, i.e., increased cell proliferation, resistance to cancer treatment, and epigenetic changes that lead to pathologic and clinical progression of cancers.[5]. The standard treatment of ovarian cancer is surgery to remove the tumor and subsequent chemotherapy . Th. Th2O2 i of GPx1 . We furt in OVCD . Althoug in OVCD , Trx1 is in OVCD . OverallBioenergetic metabolism is critical for cancer progression and aggressiveness. In this study, we compare the level of mitochondrial OXPHOS and glycolysis between OVCD with its parental cell OV90 by measuring OCR and ECAR, respectively. Figures μM -5 μM). As shown in μM of MnP induced the cell death of OV90 and OVCD by about 40%. The efficacy of MnP in killing OVCD cell line, at all tested concentrations, was superior to OV90 cell line. However, there was no decrease in cell survival of hTER7 cells at any tested concentration. This result indicates that MnP selectively kills ovarian cancer cells and spares normal ovarian epithelial cell. The data from 2O2. Further, H2O2 is a signaling ROS that regulates redox-sensitive protein activities that play crucial roles in cell growth and proliferation (at low concentration), as well as cell death (at high concentration) [2O2 is a crucial molecule by which MnP mediates ovarian cancer cell death. By measuring the intracellular H2O2, we found that MnP induced a significantly higher amount of H2O2 in OV90 cell line as well as OVCD cell line, but not in hTER7 cell line when comparing to the non-treated group. Intracellular levels of H2O2 after MnP treatment in OVCD cell line were significantly greater than in OV90 cell line  and low availability of H2O2-removing enzymes [To examine the anticancer efficacy of MnP to chemoresistant ovarian cancer cells versus toxicity to noncancer ovarian epithelial cells, we compare % cell viability of hTER7, OV90, and OVCD in response to MnP (0.033 tration) . Therefoell line . Extracereatment . To inve enzymes .μM or cisplatin in the range of 0.5 to 1 μM induced cell death of TOV21G cells at ~50% and 60%, respectively. A combination of MnP with carboplatin or cisplatin showed that MnP synergistically enhanced carboplatin and cisplatin cytotoxicity, which decreased TOV21G cell viability to around 20%.Since MnP had anticancer effect on OV90 and OVCD, we compare the efficacy of MnP with the first-line antiovarian cancer drugs, carboplatin (CBCDA), and explored the effect of the cotreatment of MnP with carboplatin. As shown in , but the accumulation of MnP, when used jointly with carboplatin, slightly decreased in OV90 and profoundly decreased in OVCD cells.To compare the accumulation of MnP, carboplatin, or the combination of MnP and carboplatin between OV90 and OVCD, we measured the drug concentration in the cells after one-hour treatment. As shown in , but the2O2 production that would cause cell death. By measuring the overall reactive species in OV90 cell line and OVCD cell line, we found that after treatment with MnP or MnP/carboplatin, MnP induced significantly higher levels of ROS/RNS generation comparing to the nontreated control. This is presumably due to the cycling of MnP with cellular reductants, most so ascorbate, which gives rise to the production of superoxide (and its progeny) and subsequently H2O2 [2O2. Although intracellular H2O2 levels in OV90 and OVCD cells were slightly but not significantly increased after MnP treatment, the levels were increased significantly with MnP/carboplatin versus control . Interestingly, MnP significantly increased extracellular H2O2 in OV90 cells and OVCD cells within the first 6 hours of treatment. Combined treatment with MnP and carboplatin further increased extracellular H2O2. The huge increase in extracellular H2O2 occurred as early as 5 minutes after MnP treatment and was inhibited by 1000 units catalase  instrument. MnP treatment greatly diminished maximum respiration and spare respiratory capacity of both OV90 cell line and OVCD cell line, whereas MnP/carboplatin greatly diminished maximum respiration and spare respiratory capacity only for OVCD cell line . On the contrary, MnP/carboplatin treatment significantly decreased glycolytic activity and glycolytic capacity in OVCD cells . The ATP production was measured after 24 hr of treatment, and the data demonstrate that MnP/carboplatin significantly decreased ATP production in both OV90 cells and OVCD cells  dioxo species which in turn oxidatively damages proteins [2O2 levels are further increased and in turn damage to cellular proteins is increased, which suppresses further cellular viability and as anticipated more so of OVCD cell line than of OV90 cell line [2O2 production using porphyrin (cationic photosensitizer) in photodynamic therapy is prone to activate cytotoxic activity of human ovarian cancer cell lines such as HeyA8MDR 35026603 and HeyA8 when compared to mouse embryonic fibroblast cell [Our study further supports this concept with the higher emphasis on the efficacy of MnP in treating chemoresistant ovarian cancers as a single drug or combined with chemotherapy. We explain the impressive efficacy of MnP, when used as a single agent, as follows. MnP accumulates in cancer cell to a high degree and it cycles there with endogenous cellular reductants whereby producing Hx levels . In a suproteins . When Mnell line –61. Simiast cell . These s2O2 activate Nrf2 via Keap1 oxidation and simultaneous interaction of Trx1 and Nrf2, leading to the enhancement of Nrf2 transcriptional activity [Keap1 is a redox-sensitive proximal regulatory protein of Nrf2. Under normal condition, Keap1 ensured low level of Nrf2 by mediating Nrf2 ubiquitination and degradation. Trx1 is a redox-sensitive protein that interacts with other proteins, including Keap1 and Nrf2, and provides electrons through thiol-disulfide exchange to maintain a reduced state and support activities of the proteins . In the activity , 63. Witactivity . In contactivity .5+, prefer accumulation in mitochondria [2O2 (originated from carboplatin) which in turn further oxidatively damages cellular proteins.Aside from the pharmacodynamics of MnP, the cellular pharmacokinetics of the drug is another factor that contributes to the differential effects of MnP on chemoresistant and chemo-sensitive ovarian cancer cells , 65. In chondria  and the chondria . Thus, i2O2 acts as signaling molecule for inducing cell death when MnP was used as a single drug or when combined with carboplatin. The upregulation of mitochondrial OXPHOS and rewired redox state in ovarian cancer cells could be used as a marker for MnP antitumor efficacy and should be considered for tailoring optimal treatment for the individual cancer patient. Thus, the redox-active MnP-based compounds affect the chemoresistant ovarian cancer cells by upregulating mitochondrial respiration and oxidative stress.In conclusion, we showed herein that MnP is highly able to induce cytotoxicity in ovarian OV90 cell line and its resistant analog OVCD cell line at very low nM levels. H


The present study aimed to optimize a liposomal formulation co-encapsulating simvastatin (SIM) and doxorubicin (DOX) that has future perspectives in anticancer therapy. The optimization process was performed by implementing the Quality by Design concept and by considering the results of a previous screening study. Failure Mode and Effects Analysis was used for the identification of the potential critical factors, i.e., phospholipid, SIM and DOX concentration, which were assessed in an optimization experimental design with the purpose of designing an optimal formulation. The optimal formulation, meeting the established quality profile, was additionally characterized in terms of the release profile and antiproliferative effects. During dissolution studies, a novel chronoamperometric method was used for the simultaneous quantification of SIM and DOX. The obtained data confirmed the similarity of this method with a validated HPLC method. The anticancer potential of the optimal formulation was tested against two human cancerous cell lines, namely T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line. The results highlighted that the antiproliferative effect of the optimal formulation is concentration dependent and favors a synergistic effect of the two drugs. The increased number of cancer cases and the negative predictions of cancer mortality are urging the scientific community to develop a better understanding of cancer biology and to develop new treatment options . One of At present, the anticancer therapies are varied, from classical chemotherapeutic drugs to immunotherapy . NeverthTo avoid drug resistance, anticancer therapeutic schemes are recommending the use of two or more chemotherapeutic drugs with synergistic effect to target multiple intracellular pathways, which will ultimately lead to the inhibition of tumor progression ,10. In tDrug development studies usually involve the use of large amounts of financial resources and time . For thiConsidering these, this study aimed to optimize a liposomal formulation co-encapsulating SIM and DOX using the QbD approach, and to perform a complete in vitro characterization of the optimal formulation. The optimization study took into account the results we obtained in the screening study of the same liposomal formulation . More prUsually, the implementation of the QbD approach starts by defining the QTPP and ends with establishing the control strategy . SettingAccording to a World Health Organization report, the first three most prevalent types of cancer in 2020 were breast, lung and colon and rectum cancer, accounting for more than three million deaths worldwide . This faYet, a liposomal formulation can be considered suitable for in vivo administration only if the quality attributes of liposomes, i.e., size, PdI, zeta potential, are attaining the target values established by the scientific community. More precisely, the liposomes should be in the size range of 50 to 200 nm and monodispersed (PdI < 0.2) to guarantee the permeation and retention of the nanoparticles in the tumor, and to avoid their uptake by the macrophages . On the The next step in the QbD approach is performing risk assessment, which has the role of identifying and quantifying the potential risk that endangers the achievement of the QTPP . AccordiCompared to the previous results , the risConsidering these results, the concentrations of PL, SIM and DOX were studied as independent variables in an optimization DoE, while the entrapped drug concentration, EE%, liposomal size, PdI, and zeta potential were the dependent variables. The composition of the formulations and the experimental results of the DoE are presented in The experimental results were fitted through multilinear regression model and the statistical parameters of the fitting model and Analysis of Variance (ANOVA) test are presented in p > 0.1).The statistical parameters of the summary of fit evidenced that the results fit well with the proposed model. However, the ANOVA statistical parameters showed that the formulation factors had a great impact on the responses, and there is no lack of fit for either of the responses  of DPPC, (ii) to the use of a higher concentration of cholesterol and PL, and (iii) to the temperature used for the release study [The release profile of liposomes is equally accountable for achieving a favorable outcome in cancer therapy, along with the entrapped drug concentration, particle size, PdI or zeta potential, the main objective being to prevent drug leakage during blood circulation time and favor the release at the tumor site ,42. As pse study ,44,45,46m of DPPC decreases when high concentrations of SIM are used, and liposomes are converted from a rigid and stable form to a more fluid and unstable form [According to the literature, DOX sulfate has reduced solubility in aqueous media , but Rusble form , which cble form . Despiteble form ,49. Thisble form , leading2 > 0.95). This result highlights that DOX is released slowly from the optimal formulation as a result of the slow dissolution process of DOX sulfate crystals and the diffusion of DOX molecules through the lipid bilayer  [2 > 0.95). This suggests that SIM, like DOX, is released slowly from the optimal formulation, but in this case, the drug release does not follow the Fick’s diffusion laws . More precisely, the release of SIM is caused to the same extent by the diffusion of SIM molecules through the lipid bilayer and by the “erosion” or “disintegration” of the liposomes’ membrane [In addition, it can be inferred from as 0.22) ,52. On tmembrane ,53.Despite the promising results that have been obtained in recent decades regarding the use of liposomes in cancer therapy, there is still some controversy around this method . In thisAs a means to better understand the pharmacokinetic profile of drugs/nanoparticles while providing accessible devices to scientists and patients to quantify different compounds from simple/complex matrices rapidly and in an easy manner, the electrochemical analysis contributes to this field with a wide variety of sensors compatible with portable devices, i.e., mobile phones, with applications in research, industry or even medical units ,57,58.Taking into account the applicability and advantages of the electrochemical techniques, we have used a chronoamperometric method developed and optimized by Rus et al.  to simulp value of the paired t-test, which was 0.13 for DOX and 0.49 for SIM, suggesting that there is no significant difference in results when the two analytical techniques were compared.To obtain a detailed understanding of these results, the Bland–Altman plot was used to evaluate the agreement between the two analytical techniques . The HPL® and Caelyx [Investigating the stability of liposomes is considered to be a critical issue in the pharmaceutical development process because the intended effect depends on maintaining the liposomes’ CQAs in the target ranges during storage ,61,62. Pd Caelyx ,68, two m produces liposomes with increased stability, while the use of a low temperature (<Tm) to store the liposomal formulations minimizes lipid mobility, thus preventing drug leakage and liposomal coalescence [Under these experimental conditions, the SIM-DOX-LCL optimal formulation presented good stability over the tested period. The % of DOX retained A was conlescence ,64. Addilescence ,69. On tlescence ,71. Becalescence . Previoulescence , and an lescence . TherefoThe other referenced CQAs, namely particle size, PdI and zeta potential, were preserved in the previously established target ranges, as can be seen in 50  were assessed in conjunction with the treatments to exclude their influence on cell viability. The results highlighted that cell viability for E-LCL was 99% (data not presented), and all the reported results exclusively represent the influence of the treatments.The long circulating liposomes loaded with DOX (DOX-LCL) were observed to increase the IC release ,74. In t50 value increasing 1.5 times for liposomes compared to free DOX. This result is in agreement with another study, which demonstrated that the T47D-KBluc cell line is more resistant to SIM therapy than other breast cancer cell lines, and higher concentrations are required to achieve an antiproliferative effect [50 by 30 times compared to free SIM, and 16 times compared to SIM-LCL.Apart from these results, SIM-DOX-LCL optimal formulation also documented increased resistance in the T47D-KBluc human mammary ductal carcinoma cell line when low concentrations of SIM (7.5–0.2 µM) and DOX 0.75–0.02 µM) were used A, the DO–0.02 µM 50 value of DOX decreased 2.5 times compared to free DOX, and 6.8 times compared with DOX-LCL. In this respect, Aksoy and Ceylan demonstrated that statins have a different impact on A549 human pulmonary cancer cell line. More precisely, they reported that high concentrations of SIM determine an increase in cholesterol concentration in the cell membrane, with a direct impact on its fluidity and permeability [50 value compared to free SIM or SIM-LCL.In contrast with the previous results, the SIM-DOX-LCL optimal formulation highlighted a higher level of activity against the A549 human pulmonary cancer cell line when smaller concentrations of SIM (7.5–0.2 µM) and DOX (0.75–0.02 µM) were used A. The ICeability . BearingThe results obtained in this study suggest that SIM enhances the anticancer properties of DOX in a free form and co-encapsulated in liposomes, achieving a synergistic effect on both studied human cancer cell lines. However, it must be taken into consideration that the antiproliferative potential of SIM-DOX-LCL is concentration dependent, and a thorough analysis should be performed before establishing the dosing regimens for the future in vivo studies. In addition to these, additional studies are required to establish the pharmacokinetic profile of SIM-DOX-LCL optimal formulation as a means to ensure the desired drug ratio at the tumor site.TM, from ThermoFisher Scientific ; resazurin and fetal bovine serum (FBS) were from Sigma Aldrich .Doxorubicin hydrochloride (DOX) from Merck KGaA , simvastatin (SIM) from Biocon Limited , 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N-(carbonyl-methoxypolyethylenglycol-2000)-1,2-distearoylsn-glycero-3-phosphoethanolamine , from Lipoid GmbH ; cholesterol (CHO) from sheep wool, resazurin and fetal bovine serum, from Merck KGaA ; sodium chloride and ammonium sulfate (AS), from Chemical Company . For cell culture assay, Dulbecco’s modified Eagle medium (DMEM) and phosphate-buffered saline (PBS) were purchased from Gibco , RPMI 1640 medium with GlutaMAXAll the other solvents and reagents were of analytic grade purity, and were commercially available.According to the ICH guideline for pharmaceutical development Q8(R2), the implementation of the QbD approach in the development of pharmaceutical formulations/products has to follow a specific number of steps, which include defining the QTPP, identifying the CQAs, interconnecting the material attributes/process parameters with the CQAs, and defining the design space . The firThe factors that counted the highest RPN were included in a DoE conceived in Modde 13 software . The CQAs that constituted the matrix of dependent variables were the entrapped drug concentration, EE%, liposomal size, PdI and zeta potential. The DoE was a central composite face design, quadratic model, with 17 experiments of which three were replicates. Modde 13 software was also used for the statistical analysis of the data and to define the design space. The optimal formulation was prepared in triplicate, the results being expressed as average ± standard deviation. For each response, the experimental value was compared to the predicted value, and the residual value was calculated using the following formula:SIM-LCL were prepared using a modified method of the lipid film hydration technique reported by Porfire et al. . In thisThe encapsulation of DOX into SIM-LCL was performed using a modified process of the active loading method with AS ,78,79. TThe preparation of DOX-LCL was performed following the techniques described at the preparation of SIM-LCL and SIM-DOX-LCL, but without the addition of SIM in the lipid bilayer.The preparation of E-LCL was performed following the technique described at the preparation of SIM-LCL, but without the addition of SIM in the lipid bilayer.The SIM and DOX entrapped concentrations were determined using an HPLC method developed and validated by Barbalata et al. . FirstlyThe EE% for SIM and DOX was determined using the formula:v/v). The results were expressed as an average.For the measurement of the liposomal size and PdI, the dynamic light scattering technique was used, while for zeta potential, the laser Doppler electrophoresis was applied. All the measurements were performed in triplicate at room temperature with a Zetasizer Nano ZS90 analyzer  after diluting the samples with double distilled water  for 72 h ,49. At pThe HPLC analysis was performed according to the previously mentioned method, while chronoamperometry analysis was carried out via a method reported by Rus et al.  and adapThe stability study was performed on the optimal SIM-DOX-LCL formulation for 28 days. The liposomes were kept at 4–8 °C and at predefined periods, samples were withdrawn and analyzed in triplicate with respect to the entrapped drug concentration (expressed as % of drug retained), EE%, liposomal size, PdI and zeta potential.The % of drug retained was calculated using the following equation:The results were expressed as average ± standard deviation.2 humidified atmosphere. The medium was changed every 2–3 days and the cells were subcultured once they reached 70–80% confluence.In this study, two cancerous cell lines were used: T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line . Cells were cultured in the recommended medium containing 10% inactive FBS and were kept at 37 °C in a 5% CO4 cells/mL for A549 cell line and 2 × 105 cells/mL for T47D-KBluc cell line) and were left for 24 h to attach to the plate. Subsequently, the cell lines were exposed to different treatments for 48 h, as shown in Prior to any treatment exposure, cells were seeded in 96-well plates  was read using a Synergy 2 Multi-Mode Microplate Reader. The experiments were performed in triplicate. The results were expressed as % of cell viability compared to the control.Cell viability has been assessed using a resazurin-based test. For this purpose, cells have been incubated with a resazurin solution of 200 µM for 4 h at 37 °C in a 5% COThe statistical analysis of the DoE was performed using Modde 13 software , while the data that resulted from the antiproliferative studies was analyzed using SigmaPlot 11 software . The ANOVA test was used to evaluate the differences between the investigated factors and the obtained results at 95% confidence level. The release profile of SIM and DOX were fitted through multiple kinetic models using DDSolver add-ins software.The process knowledge gained from the screening and optimization study of SIM-DOX-LCL contributed to the development of an optimal formulation that meets the QTPP, which was defined in conformity with the scientific literature and international guidelines. Through this study, it was evidenced that the most critical factors responsible for achieving the quality profile of SIM-DOX-LCL are PL, SIM and DOX concentration.The in vitro characterization of the optimal formulation  recommends SIM-DOX-LCL for future in vivo animal studies, since the drug combination presents a synergistic effect on both T47D-KBluc human mammary ductal carcinoma cell line and A549 human pulmonary cancer cell line, and the liposomal formulation presents a prolonged drug release. However, special consideration should be given to the administered dose of the liposomal formulation, since different concentrations of the same formulations had an opposite antiproliferative effect on the tested cancer cell lines. The applied chronoamperometric method for the simultaneous quantification of SIM and DOX evidenced the ability to quantify both APIs at low concentration levels in the release medium, but additional optimization studies are required to increase its selectivity before using complex matrices for therapeutic drug monitoring in animal models and pharmacokinetic studies.Taking these positive results into consideration, the research group intends to carry out additional studies concerning the pharmacokinetic profile, antitumor efficacy or toxicity profile, to establish the in vivo benefits of associating SIM and DOX in a liposomal formulation.


After confirming that the 4-amino group was not part of the active pharmacophore, chalcones were attached to transferrin-conjugated carbon-dots. These conjugates were significantly more cytotoxic than the free chalcones, with the C-dot-transferrin-2,5, dimethoxy chalcone conjugate inducing up to 100-fold more GSC death. Several of the tested chalcones represent promising lead compounds for the development of novel anti-GSC drugs. Furthermore, designing amino chalcones for carbon-dot mediated drug delivery is a rational and effective methodology.The current prognosis for glioblastoma is dismal. Treatment-resistant glioblastoma stem cells (GSCs) and the failure of most drugs to reach therapeutic levels within the tumor remain formidable obstacles to successful treatment. Chalcones are aromatic ketones demonstrated to reduce malignant properties in cancers including glioblastoma. Nanomedicines can increase drug accumulation and tumor cell death. Carbon-dots are promising nanocarriers that can be easily functionalized with tumor-targeting ligands and anti-cancer drugs. Therefore, we synthesized a series of 4′-amino chalcones with the rationale that the amino group would serve as a “handle” to facilitate covalent attachment to carbon-dots and tested their cytotoxicity toward GSCs. We generated 31 chalcones (22 4′-amino and 9 4′ derivatives) including 5 novel chalcones, and found that 13 had an IC The prognosis for individuals diagnosed with Glioblastoma (GBM), the most common and lethal primary brain tumor in adults, remains dismal. Even with an aggressive standard of care that includes; maximal safe tumor resection, radiation therapy with concomitant chemotherapy (temozolomide), the average survival is only 14–15 months [Although GSCs, referred to as GBM stem-like cells or tumor initiating cells, represent only a small population of cells within the tumor, they have the capacity to divide asymmetrically, giving rise to additional GSCs as well as the more differentiated cells that make up the bulk of the tumor . EmerginNanoparticle-mediated drug delivery has the potential to cross the BBB and increase drug concentrations within the tumor and therefore increase treatment efficacy. To date most anti-cancer nanomedicines evaluated in clinical trials ,13 rely 2 group that is not readily available on most anti-cancer drugs. To overcome this obstacle, we synthesized a series of 4′-amino chalcones with the idea that the amino group would serve as a “handle” for conjugation to C-dots.Non-toxic carbon-dots (C-dots) have emerged as excellent choices for developing drug delivery systems (DDS) and bioimaging agents due to their high biocompatibility, nano-size (<10 nm), photoluminescent properties, and versatility of surface functionality . DerivedChalcones are aromatic ketones belonging to the family of polyphenolic compounds. The anti-cancer properties of various natural polyphenolic compounds are well known ,24,25, aTherefore, the objective of this work was to synthesize a series of 4′-amino chalcones, test their cytotoxicity against a panel of GCS lines, and to investigate the potential of using C-dots as a nanocarrier to increase the anti-cancer effect. To study the role of the 4′-amino group in binding interactions (electrostatic vs. H-bonding), we synthesized N-acyl derivatives and 6-hydroxy derivatives of some of the more potent 4′-amino chalcones and tested the cytotoxicity in our cell lines. Lastly, we generated transferrin tethered C-dot-chalcone conjugates to investigate the enhancement of chalcone therapeutic efficacy by docking them into the tumor cell via the receptor mediated endocytosis. 1H- and 13C-NMR spectra were recorded at 500 and 125 MHz on Bruker. The spectra were referenced to the residual protonated solvents. Abbreviations such as s, d, t, m, br, and dd used in the description denote singlet, doublet, triplet, multiplet, broad, and double doublet, respectively. The chemical shifts and coupling constants were reported in δ parts per million (ppm) and hertz (Hz), respectively. High resolution mass spectra were obtained on Bruker micrOTO-Q II mass spectrometer. All intermediate and final products were monitored by thin layer chromatography (TLC) on 250 μm silica plates. Where applicable, the compounds were recrystallized from the proper solvent or purified by flash column chromatography on silica gel (200–300 mesh) with ethyl acetate/hexanes (1:1) as eluant. All reagents were obtained from commercial sources and were used without further purification. 3a–3x, 4′-hydroxychalcones 4a–4f and 3′-aminochalcone 5a are outlined in 3a , 3b , 3c , 3e , 3f , 3i , 3k , 3l , 3n , 3o , 3p , 3q , 3r , 3s , 3t , 3u , 3v , 3w , and 3x , from the 4′-amino series; chalcones 4a , 4b , 4c , 4d , 4e and 4f , from the 4′-hydroxy series; and chalcone 5a . MOM-protected derivative of vanillin was synthesized according to literature procedures [The syntheses of 4′-aminochalcones ocedures . 2 incubator.The generation and characterization of our GSC lines  have been previously described ,51,52. IThe chalcones were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mM, vortexed and subsequently diluted to obtain 1 mM and 0.1 mM stock concentrations. C-dot-trans-chalcones were resuspended in supplement-free media (3:1 ratio DMEM/F12 for GSC experiments or RPMI for non-stem cell line experiments). Cell viability in response to drug treatment was determined using CellTiter 96 AQueous One Solution Cell Proliferation Assay, a colorimetric MTS assay kit . The GSCs were seeded into 96-well plates using modified neurosphere media containing 5% FBS at a density of 10,000 cells per well. MSCs and U87 cells were plated at 5000 cells/well while SJ-GBM2 cells were plated at 10,000 cells/well. The next day cells were treated with increasing concentrations of chalcones or C-dot-chalcone conjugate. Next, 72 h later, media was aspirated and 100 μL of a 1:5 solution of MTS to cell culture media was added to each well and incubated for 1–4 h. Optical density was measured at 490 nm using BioTek Synergy HT plate reader. Viability of drug treated cells is expressed as the percent viable cells relative to non-treated cells (100% viability). Experiments were done in triplicate.The effect of chalcones’ clonogenic growth potential was determined using sphere-forming assays. Glio3 and Glio38 neurosphere cell lines ,7 were dOur Western blot methods have previously been described ,22,51,52N-hydroxysuccinimide (NHS) (10.68 mg/mL in PBS) was added, and after 20 min, 1 mL of transferrin (3 mg/mL in PBS) solution was added; 45 min later, 4.80 or 4.38 mg in 1 mL of 3,5-dimethoxy chalcone (3f) or 3,4,5-trimethoxy chalcone (3e) in DMSO was added to the C-dots reaction mixtures, respectively, and stirred overnight at room temperature. The mixtures were dialyzed using a 3500 molecular weight-cutoff dialysis membrane for 4 days, replacing the deionized water every 4–10 h. The reaction mixture was further purified by size-exclusion chromatography. The resultant eluent was collected into different aliquots and analyzed by ultraviolet–visible spectroscopy (UV-Vis) and fluorescence spectroscopies to identify the C-dots-trans-chalcone derivatives. The conjugates were frozen at −80 °C for 24 h and then lyophilized for 3 days to acquire the powdered sample.C-dots were synthesized from carbon-ash using an acid-oxidative top-down method commonly used in our group as previously described ,55. BrieThe conjugates (20 μg/mL) were characterized by UV-Vis spectroscopy in a 1 cm quartz cell using Shimadzu UV-2600 spectrometer. The fluorescent emission spectra of the C-dots-trans-chalcone conjugates and the free chalcone were obtained by Horiba Jobin Yvon Fluorolog-3 with slit width 5 nm for both excitation and emission. The molecular weights of each C-dot-trans-chalcone derivative was analyzed with a Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) mass spectrometer using a Bruker auto flex speed spectrometer.t-tests for all pairwise comparisons of the different treatments that were tested. The results are presented as the mean  ±  standard error mean (SEM). Significance was set at p  <  0.05. Significance was determined by one-way analysis of variance (ANOVA) when comparing results of multiple chalcones together.Significance was determined using Student’s 3a–3x, 4a–4f and 5a was performed according to 3a–3x) were synthesized by Claisen–Schmidt reaction with yields of 27–97% after chromatography purification. The series of compounds has chalcones with amino at position 4′ (ring A) and benzene ring B substituted by electron-withdrawing (EWG) and electron-donating groups (EDG). Additionally, aryl analogues with benzene ring B replaced for furan and pyridine rings were synthesized  with acetyl chloride and triethyl amine. Subsequently, another series of chalcone analogues (4a–4f) were synthesized. These analogues were prepared by a base-catalyzed Claisen–Schmidt condensation between 4′-hydroxyacetophenone and the appropriate aryl aldehyde with yields of 82% to quantitative after purification. The 4′-O-acetyl analogue (4d) was prepared by condensation of chalcone 4c with acetyl chloride in the presence of triethylamine. Analogue 4e was prepared by reacting chalcone 4c with methyl bromoacetate under basic conditions. Hydrolysis of 4e under basic conditions (LiOH/THF/MeOH/water) followed by acidification afforded the acid 4f. More detailed description of the synthesis along with the spectral data for each compound can be found in the experimental section of the The synthesis of two series of chalcone derivatives thesized . Chalcon50s for the 4′-aminoacetophenone series is shown in 3d, 3f, 3g, 3o, 3p, 3q, 3u, 3v, 3x) had IC50 values below 10 µM in all GSC lines. Although chalcones 3c, 3e and 3w were not as active, treatment of the GSCs did result in IC50 below 15 µM across all three cell lines. In general, these chalcones were relatively non-toxic to non-cancerous mesenchymal stem cells (MSCs) with a few exceptions. Notably, chalcones 3g and 3p demonstrated similar toxicity to both GSCs and MSCs. The relative IC3u, 3v, 3w, 3x). Due to the potential for multiple interactions with proteins, halogens have found applications in medicinal chemistry [50 for the 3 GSC was 8.6 µM, 7.2 µM, 9.1 µM, and 7.4 µM for chalcones 3u, 3v, 3w, and 3x, respectively. Interestingly distinct differences were observed between the different GSC lines. This is illustrated in Some of the more effective chalcones were those containing halogens: chlorine, bromine or fluorine  and pyridine  chalcone derivatives. Of these, only the pyridine derivatives were effective. While the position of the nitrogen seemed to play a minor role in the cytotoxicity toward GSCs, the average IC50 across all three cell lines of 6.1 ± 0.4 µM for the nitrogen at the 2 position (3p) and 7.2 ± 0.5 µM for the nitrogen at the 3 position (3q), the toxicity to normal non-cancer MSCs was highly different. Chalcone 3p gave an estimated MSC IC50 of 8 µM compared to an estimated MSC IC50 of well above 10 µM for chalcone 3q. These data indicate that 3q rather than 3p would be a better lead compound for further development. We investigated both furan , antitubercular , and anti-cancer  . Howeverntration . This su3m), the 3-methoxy-4-methoxymethoxy (3h), 4′-amino-3 ethoxy-4-hydroxy (3j) as well as 2 N-acetylamino derivatives (3d and 3g). Anti-cancer drugs containing morpholine interacting moieties include inhibitors of the PI3K/Akt/mTOR signaling pathway, perhaps one of the most commonly altered signaling pathways in cancer [3n) was also inactive at the concentrations tested. The 4′-3-ethoxy-4-hydroxy chalcone (3j) was not active either at the concentrations tested. Based on our data, the presence of a hydroxy group at position 4 also appears to be deleterious for activity. Chalcones 3i, 3j, 3k and 3h all contain a hydroxy group at position 4 and were not active at concentrations tested. This is best illustrated by looking at chalcones 4e, 4f and 4k. The 3,4,5-trimethoxy chalcone (4e) was active as was the 3,5-dimethoxy chalcone (4f) suggesting that the methoxy or any group at position 4 is not necessary for activity. However, substituting the methoxy group with a hydroxy group at position 4 (4k) eliminates activity. Both novel N-acetylamino chalcones, the 2,5-dimethoxy (3d) and the 3,5-dimethoxy (3g) were active and will be discussed in greater detail in the next section. However, it is important to note that while 3d was only moderately toxic to MSCs with an IC50 of 16.8 µM, 3g induced significant MSC death with an IC50 of 7.4 µM. We also synthesized five novel chalcones; three 4′-amino derivatives; 4-morpholino , whereas the 2,3 and 3,4  were less effective, suggesting that the methoxy at position 5 is important for activity. Furthermore, the novel N-acetylamino 2,5 and 3,5-dimethoxy chalcone derivatives  were even more effective, indicating that the 4′ amino group is not involved in electrostatic interactions. In addition, this may suggest that a longer group at the 4′ position is increasing the interaction or strength of the drug with the binding pocket of the therapeutic target. As shown in 50 for the amino derivatives  is significantly higher than that of the acetylamino . To explore the effects of a better hydrogen-bond donor, we synthesized a series of 4′-hydroxy chalcones.In our previous work, we determined GSC cytotoxicity of curcumin-inspired bis-chalcones and found that methoxy groups were important for activity, with the greatest activity observed in the trimethoxy analog . Therefo50 for the 3′-amino trimethoxy chalcone and the 4′-hydroxyacetophenone series is shown in 4b, 4c and 4d) out of the 6 4′-hydroxyacetophenone chalcones tested had IC50 values below 10 µM. Furthermore, these were not as toxic to the non-cancer stem cell line. Conversely, the 3′-amino trimethoxy chalcone induced robust cell death in all 3 GSC lines as well as the MSC line. The IC4a) was more effective than the 4′-amino-2,3-dimethoxy (3a). Similarly, the 4′-hydroxy 3,5-dimethoxy analogue (4b) was more effective than the amino-3,5-dimethoxy (3f), which is illustrated in 4c), 4′-acetoxy (4d) and 3′-amino (5a) were more effective than the 4′-amino trimethoxy (3e), which is illustrated in 50). Taken together, these data imply that the 4′-amino group is not required to interact via electrostatic attractions for activity and these modifications of the 4′-amino group or moving the amino group actually increases the efficacy of the chalcone. It is important to note that while moving the amino group to the 3′ position increased the toxicity to GSCs, it also dramatically increased the cytotoxicity to the non-cancer stem cells, MSCs. The MSC estimated IC50 for the 4′-amino-trimethoxy chalcone was close to 50 µM compared to an IC50 of 5.5 µM for the 3′-amino-trimethoxy chalcone. Substitution of the amino group with larger groups methoxycarboxymethyl and carboxylicmethyl (4e and 4f) decreased the cytotoxicity towards GSCs possibly by altering either the binding strength, electrostatic repulsion (carboxylate group), or the target of the trimethoxy derivative. The 4′-hydroxy-2,3-dimethoxy analogue  is a rule of thumb used in the development of drug-likeness . The phynds ≤ 10 . HoweverSC lines . We incl3e) and, the 3,5 dimethoxy (3f) chalcones to conjugate with C-dots as these promoted GSC death yet were relatively non-toxic to MSCs. The C-dots were conjugated to transferrin (trans) and to chalcones via covalent amide linkage  conjugate was more effective than the trimethoxy (3e) conjugate for all cell lines. We previously demonstrated transferrin receptor expression in the GBM non-stem cell lines [To determine if the C-dot-trans-chalcone conjugate could induce GSC death, cells were treated with increasing concentration ranging from 0.01 to 0.5 µM and resulting viability determined 72 h later. Prior to in vitro studies, C-dot-trans-chalcone conjugates were resuspended in cell culture media and a 0.1 mM stock was prepared based on the molecular weight of each, which is 85,000 g/mol. As shown in ll lines ,20. Simi50 for the free trimethoxy (3e) was 9.7 ± 0.8 µM, the average GSC IC50 for C-dot-trans-3e conjugate was 0.42 ± 0.03 µM, representing about a 25-fold increase in GSC mediated cell death. The dimethoxy conjugate was even more effective, with an average GSC IC50 for the free dimethoxy (3f) of 8.2 ± 0.8 µM and the average GSC IC50 of 0.09 ± 0.02 µM for C-dot-trans-3f conjugate, indicating an approximate 100-fold increase in efficacy over drug alone  and SJGBM-2 (pediatric). As shown in 50 values of 3,5 dimethoxy chalcone (3f) for U87 and SJGBM2 cells were 8.5 ± 0.04 µM and 7.3 + 0.2 µM, respectively, while the IC50 values of C-dot-trans-3f for U87 and SJGBM2 were 0.12 ± 0.005 µM and 0.11 ± 0.009 µM, respectively. The fact that these values are similar to those observed for the GSC lines, suggests that both the free chalcone and the C-dot-trans conjugate can target both the GBM stem cell and non-stem cell population. Overall, the C-dot-trans-3f conjugate was about 75-fold more effective than the free chalcone in the non-stem cell lines, U87 and SJ-GBM2. However, both C-dot conjugates were significantly more effective than the free chalcone indicating that targeted C-dot mediated chalcone delivery increases its toxicity A. While ug alone A. Subseq3f on GSC self-renewal; sphere-forming assays were performed. Glio3 and Glio38 cells were treated with c-dots alone, transferrin alone or 10, 50 and 100 nM of the C-dot-trans-3f conjugate and the number of neurospheres counted 10 days later. As shown in To determine the effect of C-dot-trans conjugate Despite extensive research aimed at understanding the molecular drivers of GBM as well as thousands of clinical trials worldwide, there has been little improvement in patient outcome in decades. In recent years, it has become evident that treatment resistant GSCs contribute to the poor outcome in GBM. GSCs drive tumor growth, contribute to tumor heterogeneity and are responsible for tumor recurrence. Targeting these cells has become a major area of research. 50 values of about 10 µM or below in all 3 GSC lines. Furthermore, several of these chalcones were relatively non-toxic to non-cancerous human MSCs. In general, the halogen containing chalcones  demonstrated good activity against the GSCs with only moderate toxicity toward MSCs. Of the heteroaryl chalcones, the pyridines  rather than the furans  were more active, however the position of the nitrogen greatly affected the toxicity toward the MSCs. The most effective chalcone synthesized was the nitro chalcone (3o), which has an average GSC IC50 of about 6 µM. Furthermore, the nitro chalcone was the only chalcone to significantly reduce GSC neurosphere formation at sub-toxic concentrations, indicating the ability to target GSC stem cell self-renewal pathways. Lastly, the nitro chalcone was minimally toxic to normal cells, with an MSC IC50 of 41 µM. Methoxy containing chalcones were also effective against GSCs with the following caveats. The 3,4,5-trimethoxy (3e) was effective, however, substituting a hydroxy at position 4 abrogated activity (3k). For the dimethoxy chalcones, the position of the methoxy groups is important for activity, with the 2,5 and 3,5  dimethoxy chalcones being more effective than the 2,3 or 3,4 , suggesting that the methoxy at the 5 position is important for activity. The N-acetylamino 2,5 and 3,5-dimethoxy chalcone derivatives  were more effective than their 4′-amino counterparts  indicating that the 4′-amino group is not involved in electrostatic interactions. Similarly, the 4′-hydoxy derivatives  were more effective than their 4′-amino counterparts . However, the hydroxy and N-acetylamino derivatives were also more toxic to MSCs. Taken together, these data suggest that the 4′-amino group is not required to interact via electrostatic attractions for activity. Using the amino group as a handle for conjugation we then conjugated two chalcones  to transferrin tethered C-dots via the amide linkage and demonstrated that this C-dot DDS was up to 100-fold more efficacious than the free chalcone in our GSC lines and in non-stem GBM cell lines (U87 and SJGBM2). Both natural and synthetic chalcones have been shown to decrease the malignant characteristics of cancer stem cells including GSCs. The aim of this study was to examine the potential of targeting GSCs using small molecule chalcones. Of the 31 chalcones synthesized and tested, 14  resulted in ICThe ease of 4′-amino chalcone synthesis and their straightforward conjugation to C-dots makes them excellent lead candidates for the development of GSC-targeted anti-GBM therapies. C-dots have emerged as an exceptional choice for a DDS especially for delivery across the BBB owing to their small size, easy functionalization, and high biocompatibility ,67,68,693d, 3g, 3h, 3j, and 3m), two of which demonstrated activity against GSCs. Overall, we identified 14, which induced robust GSC death with IC50 values of about 10 µM or below in all three GSC lines. Furthermore, many of these chalcones were relatively non-toxic to non-cancerous human MSCs. The BBB severely limits the efficacy of systemic treatment as most anti-cancer drugs cannot cross the BBB at therapeutic levels. However, the physiochemical properties of these 14 compounds suggest the ability to cross the BBB, although additional studies are required. The results suggest that several of the chalcones may serve as lead compounds for further structural optimizations to find more potent and less toxic derivatives for treatment of GBM alone or as nanoparticle formulation via covalent attachment.In summary, we designed, synthesized and evaluated three series of 31 chalcones against glioblastoma stem-like cells . Among the 31 chalcones, five were novel chalcones (


Purpose: Many natural agents have a high anticancer potential, and their combination may be advantageous for improved anticancer effects. Such agents, however, often are not water soluble and do not efficiently target cancer cells, and the kinetics of their action is poorly controlled. One way to overcome these barriers is to combine natural agents with nanoparticles. Our aim in the current study was to fabricate an anticancer nanoformulation for co-delivery of two natural agents, curcumin (CR) and colchicine (CL), with a core-shell structure. Using cancer cell lines, we compared the anticancer efficacy between the combination and a nanoformulation with CL alone. Methods: For the single-drug nanoformulation, we used phosphonate groups to functionalize mesoporous silica nanoparticles (MSNs) and loaded the MSNs with CL. Additional loading of this nanoformulation with CR achieved the co-delivery format. To create the structure with a core shell, we selected a chitosan–cellulose mixture conjugated with targeting ligands of folic acid for the coating. For evaluating anticancer and apoptosis effects, we assessed changes in important genes and proteins in apoptosis  in several cell lines . Results: Nanoformulations were successfully synthesized and contained 10.9 wt.% for the CL single-delivery version and 18.1 wt.% for the CL+CR co-delivery nanoformulation. Anticancer effects depended on treatment, cell line, and concentration. Co-delivery nanoformulations exerted anticancer effects that were significantly superior to those of single delivery or free CL or CR. Anticancer effects by cell line were in the order of HCT-116 > A549 > HOS > MCF-7. The lowest IC50 value was obtained for the nanoformulation consisting of CL and CR coated with a polymeric shell conjugated with FA . With dual delivery compared with the free agents, we detected strongly increased p53, caspase-3, and Bax expression, but inhibition of Bcl-2, suggesting promotion of apoptosis. Conclusions: Our findings, although preliminary, indicate that the proposed dual delivery nanoformulation consisting of nanocore: MSNs loaded with CL and CR and coated with a shell of chitosan–cellulose conjugated folic acid exerted strong anticancer and apoptotic effects with potent antitumor activity against HCT-116 colon cells. The effect bested CL alone. Evaluating and confirming the efficacy of co-delivery nanoformulations will require in vivo studies. A co-delivery nanosystem (CDNS) consists of nanoformulations based on nanoparticles  and two or more pharmaceutical agents for their targeted delivery to selected cells. This construct has received considerable attention recently because it offers potential advantages in the treatment of disease, including cancers ,2. Such The nanocarrier is a key factor in determining CDNS success. Mesoporous silica nanoparticles (MSNs) are inorganic-based nanocarriers and among the most investigated nanomedicine platforms as systems for drug delivery (DDSs). Features of MSNs include a high surface area, large size, and large pores, suitable for surface modifications, high loading capacity, multifunctional cancer targeting, and possible large-scale synthesis. Thus, they are an ideal nanomedicine platform and have been used in DDSs targeting cancers. Among other groups, we have published findings using several DDSs and various kinds of MSNs against cancer cells ,11,12. RCurcuma longa plant. Evidence hints that CR shows several pharmacological activities associated with good safety. Additionally, it has been identified as acting via multifunctional anticancer mechanisms, and thus might be likely to improve therapeutic effects when combined with a variety of drugs  × 100. The IC50 (concentration yielding 50% inhibition of cell viability) was calculated by applying various concentrations of treatments and the probit analysis method with a t-test .Cells were seeded in 96-well microtiter plastic plates to a density of 10® 2.1  to calculate the threshold cycle (Ct) using the second derivative maximum. After normalization to GAPDH expression, the fold-change value for each gene was calculated using 2−ΔΔCt.We profiled the expression of pro- and anti-apoptosis markers in HCT-116, HOS, A549, and MCF-7 cells exposed for 72 h to IC50 doses of the treatments. In brief, treated cells were collected and total RNA isolated per instructions . One microgram purified RNA was used to synthesize cDNA in a final reaction volume of 20 µL using the QuantiTect Reverse Transcriptase Kit . Thermal programs were conducted on a Rotor-Gene Q 5-Plex HRM thermal cycler  and PCR amplicon specificity was confirmed using QuantiTect SYBR-Green PCR Kit  as previously described and following standard protocols . Forwardg for 10 min at 4 °C. Supernatants were transferred to new tubes and the protein content quantified . For each sample, 20 µg of each protein extract was mixed with 2× loading buffer, heated at 95 °C, and separated on 12% SDS-PAGE gels. After transfer of gels, a polyvinylidene fluoride membrane, the membrane was blocked with 5% non-fat milk diluted in phosphate-buffered saline containing 0.1% Tween-20. Target proteins were immunodetected with the respective primary antibody anti-Bax E63 monoclonal, ab32503; . As secondary antibodies, we used anti-mouse IgG  and anti-rabbit IgG .Immunoblotting analysis was carried out as previously described . After 7Results of analyses of biological data are given as mean ± standard deviation (SD). Significant differences for anticancer effects were evaluated by one-way analysis of variance . Analysis of RT-PCR results was conducted by one-way ANOVA in rapid publication-ready MS Word tables according to .The XRD patterns for MSNP and nanoformulations are shown in After all of the materials were heated to ~770 °C, we observed a great change in mass loss (wt.%). The weight loss varied from 21.5 wt.% (MSNP) to 32.4 wt.% (MSNPCL), 50.5 wt.% (MSNPCLCR), and 60.9 wt.% (MSNPCLCR/CSCEFA), respectively, ascribed to the surface modification with phosphonate groups, CL loading, co-loading of CR and CL, and coating with polymer mixtures . The wei−1, which strongly confirmed the surface modification compared with MSNs [−1, a new peak at 1600 cm−1, and a peak at 3354 cm−1 that was slightly less intense compared with MSNP. Further CR loading, as indicated by MSNPCLCR, resulted in more intensive peaks at 802 and 960 cm−1, new peaks in the region between 1333 to 1743 cm−1, and broader peaks centered at 2930 cm−1. These peaks could have corresponded to the presence of loaded CR. For the changes before and after polymeric coating, −1 in MSNPCLCR were slightly lower in MSNPCLCR/CSCEFA because of the polymeric coating.FTIR-ATR spectra showed obvious changes in surface-modified nanoparticles and nanoformulations A. In MSNith MSNs . Concernp < 0.05), inhibiting all four cell line cells. MSNPCLCR and MSNPCLCR/CCFA generally had better anticancer effects than MSNPCL, which was expected. Free CR had a greater anticancer effect than free CL on all cells. As Taken together, the results indicate that, compared with free CL and CR, the nanoformulations, especially MSNPCLCR and MSNPCLCR/CCFA, showed better anticancer activity and superior killing of HCT-116 over the MCF-7, HOS, and A549 cell lines. The data also demonstrate that dual-delivery nanoformulations were efficient compared with single-delivery nanoformulation. This observation is closely connected with the lowest anticancer potential of free CL versus free CR, as indicated in all four cancer line results.To identify a potential mode of action for the growth inhibitory effects of nanoformulations and free natural agents against cancer cell lines , we examined expression of key apoptosis genes. We observed potential enhanced effects of nanoformulations compared with free CL and CR on the three cancerous cell lines after a 72 h exposure to IC50 concentrations MCF-7, . The mosTo further explore potential mechanisms, we used immunoblotting with specific antibodies against p53, caspase-3, Bax, and Bcl-2 to trace the path of the cell death program in MCF-7, HCT-116, and HOS cell lines after 72 h of incubation with treatments. The results indicated that p53, caspase-3, and Bax proteins increased in the three cell lines exposed to treatment with the IC50 of MSNPCR/CSCEFA compared with MSNPCL, MSNPCLCR, CL, and CR . Levels Improvement in the anticancer activity of natural agents is important not only to enhance safety but also to move them towards clinical application. The integration of drug delivery and nanotechnology for cancer therapy has gained considerable attention ,41,42,43The results of several analytical techniques used to characterize the physicochemical properties of nanoformulations confirm the successful formulations of the proposed dual delivery system. The morphological changes seen in TEM and STEM images confirmed the CS-CE polymer mixture coating shell in MSNPCLCR/CSCEFA. This observation agrees with our previous findings using core-shell nanoformulations with a mixture of different polymers to deliver a thymoquinone prodrug with MSNs . SimilarFTIR characterization confirmed the presence of dual CL and CR natural agents and also the polymeric coating by revealing several characteristic corresponding peaks for the nanoformulations. This observation is inconsistent with previous results for two drugs combined in MSNs, such as the dual loading of the anticancer drug pemetrexed and the natural agent ellagic acid . One impThe anticancer evaluations demonstrate that the nanoformulations significantly inhibited the proliferation of MCF-7, HCT-116, HOS, and A549 cell lines more so than did free CL and CR. The nanoformulations containing dual agents (MSNPCLCR and MSNPCLCR/CCFA) were more effective than MSNPCL . The results are in line with published data for the dual delivery system of anticancer therapies encapsulated in MSNs compared with free components ,54,57,58Apoptosis is part of different physiological and pathological responses , and theAnother important characteristic of the apoptosis pathway is p53 protein activation. The interaction of cell cycle inhibition and apoptosis is important for anticancer therapy, and p53 is crucial to tumor sensitivity to drugs ,67. The Bcl-2 inhibits programmed cell death in cancers and promotes cell survival, supporting cancer resistance to drugs ,69. Bax We describe a new co-delivery system constructed by combining CR and CL without polymeric coating  and with a CS-CE-FA polymeric coating  using MSN as drug nano-vehicles. The dual delivery nanoformulation of both core and core-shell composed of ~18.1 wt.% (CL and CR) exhibited superior anticancer effects through enhanced apoptosis induction compared with single delivery of CL with 10.9 wt.% and free natural agents tested in equivalent concentrations.Through in vitro tests with four cell lines—MCF-7 breast adenocarcinoma, HCT-116 colon cancer, HOS bone cancer, and A-549 non–small cell lung cancer—IC50 results showed that the dual delivery of MSNPCLCR and MSNPCLCR/CSCEFA was most efficient, in this order: HCT-116 > A549 > HOS > MCF-7. The lowest IC50 value was obtained for the coated nanoformulation (4.1 ± 0.05 µg/mL). Apoptosis in cancer cells improved following the dual delivery nanoformulations, with promoting expression of p53, caspase-3, and Bax and inhibiting expression of Bcl-2.The promising findings from our study, using a co-delivery system for two natural agents with MSNs, should be verified through in vivo experiments to confirm the potential therapeutic anticancer effect.


The molecular diversity was carried out using SSR markers linked to heterotic QTL and up on diversity analysis it classified selected genotypes in to three distinct groups. Among the selected inbred lines, a wider genetic variability and molecular diversity were observed. A total of 39 test crosses were generated after classifying 16 inbred lines in to three testers and thirteen lines (based on per se grain yield and molecular diversity) and crossing them in line × tester manner.The present investigation was carried out to select the best medium maturing inbred lines, among a set of 118 inbred lines. Based on the Duncan multiple range test, out of 118 lines, 16 inbred lines were selected on the basis of its high yield Combining ability analysis of these parental lines showed that female parents, PML 109, PML 110, PML 111, PML 114 and PML 116 showed additive effect for KRN and grain yield, whereas male parents, PML 46, and PML 93 showed epistatic effect for KRN and PML 102 showed epistatic effect for grain yield. The generated information in the present investigation may be exploited for heterosis breeding in filed corn.To tackle the balanced dietary requirement of Indian population; we focused to enhance the productivity of maize hybrids using genetically broad based, elite, diverse inbred lines. Combination of selection criterion, not only augment the productivity but also improves the quality of hybrid/s.The online version contains supplementary material available at 10.1007/s11033-022-07295-3. Maize is one of the key cereals, which plays the major role in Indian agriculture, especially to meet the staple food, livestock feed, edible oil and biofuel demand of growing population and industry. Hybrids play crucial role in maize productivity, which not only enhance production but also alleviate food scarcity and nutrition requirement of the developing countries. Hybrid maize cultivars development needs selection of appropriate parents (inbred lines) which is the concealed of success in hybrid maize development. Identification of high yielding hybrids require careful selection of parents based on their combining ability and underlying genetic constituents of inbred lines .1) hybrids exhibiting superior vigor for plant growth and grain yield was first exploited in maize. Though the mystery of heterosis has been explored for over a century, but the underlying mechanism remains insufficiently understood [The extent of enhancement of maize productivity not only depend on the genetic variability but also diversity of the parental inbred lines involved in the cross combination, which ultimately determine the magnitude of heterosis. The power of heterosis by breeding filial one .A set of 118 stabilized maize inbred lines, which were derived from the diverse source of population and evaluated with two rows each of genotypes along with reiterating high yielding checks at regular interval using augmented block design during 2016 and 2016–17 at ICAR-Regional Research Centre, Dharwad, Karnataka. These inbred lines were categorized into different maturity groups based on their flowering time. The Duncan multiple range test was used for selection, out of 118 inbred line, 16 top performing medium maturing inbred lines were selected based on yield http://stat.iasri.res.in/sscnarsportal).The selected 16 inbred lines were evaluated in replicated trials under Randomised Complete Block Design (RCBD) with two rows each of test genotypes at ICAR-Indian Agricultural Research Institute, New Delhi during 2017 and used for further investigation. The recommended package of practices was followed to raise a healthy crop. Data on grain yield component traits viz., cob length (CL) (cm), cob girth (CG) (mm), kernel row number (KRN) and kernel per row (KPR) were recorded along with grain yield (kg/ha) using standard methodology and data were analysed through SAS 9.3v software (www.maizegdb.org). A total of 50 linked SSR markers (Supplementary Table 2) distributed across the different chromosomes (1–10) were used for molecular diversity analysis. The genomic DNA of 16 parental lines was isolated using CTAB (Cetyl-trimethyl ammonium bromide) method [Taq DNA polymerase , 10X reaction buffer , 0.1 mM dNTPs, 10 pmol/ µL each primer and 50 ng DNA template in a total reaction volume of 25 µL. The PCR amplification was carried out with initial denaturation at 94 °C for 5 min. followed by 35 cycles consisting of denaturation at 94 °C for 30s, annealing at 55 °C for 30s, extension at 72 °C for 60s and a final extension of 7 min. at 72 °C. The PCR amplified fragments were resolved o 3.5% (w/v) agarose gel (HiMedia) and the amplified products were scored the estimated polymorphism information content (PIC) values as per Anderson et al. 1993 [Heterotic QTL based markers with high LOD value (˃ 4) were selected and primer details were collected from Maize GDB  method . The PCRosomes 1– were use° 49’ N latitude, 75° 43’ E longitude and 593 mean sea level) during 2018.The inbred lines selected in the field experiment were classified as female (13) and male (three) parental lines based on their variability and molecular diversity. A set of 39 test cross hybrids were generated using these parental lines by following line × tester mating design at ICAR-Indian Agricultural Research Institute, New Delhi, during 2017–18. The generated test cross hybrids were evaluated in RCBD with two replications at Regional Agricultural Research Station, Vijayapura , competitive plants were tagged and numbered in the middle row to observe yield and other quantitative characters. Data were recorded on CL (cm), CG (cm), KRN, KPR and grain yield (kg/ha) at respective stages of growth and development of the crop. The software, TNAUSTAT was used to estimate the combing ability and other descriptive statistics .per se grain yield recorded was 2365 kg/ha which ranged from 1087 to 3113 kg/ha (Supplementary Table-3).Analysis of variance of 16 inbred lines indicated wider variability and significant differences among each other for the trait under consideration  markers, which is linked to yield and heterotic QTLs in maize. These linked markers (50 no.s) were used for polymorphic survey using a set of 16 inbred lines to understand the molecular diversity among the lines. These markers found highly polymorphic among parental lines . Cluster I had eight inbred lines , cluster II had seven inbred lines  and the cluster III was mono-genotypic (PML 46). The clusters mean for the grain yield was 2196.40 kg/ha, 2464.06 kg/ha and 3026.25 kg/ha, for Cluster I, Cluster II and Cluster III respectively. Similarly, cluster mean for CL , for CG  and for KRN , respectively were also recorded by these clusters (Supplementary Table 5). This indicated that the inbred lines belonging to these clusters have substantial genetic diversity.The analysis of variance for morpho-physiological and yield related traits among 39 test hybrids obtained by the crossing of 13 inbred lines with three testers, showed that the mean sum of squares due to the traits studied were highly significant, indicating the presence of substantial differences among the hybrids for all the studied traits Table . VariancGeneral combining ability indicates the average performance of the lines in a series of cross combination. Among the tested lines, the PML 116 is having the significant positive GCA effect for cob girth (0.61) followed by PML 110 (0.59), PML 111 (0.57), PML 114 (0.53) and PML 109 (0.46). For the KRN, the lines PML 116 is having the significant positive GCA effect (1.28) followed by PML 110 (1.15), PML 111 (1.01), PML 109 (0.85) and PML 114 (0.82). Hence, these are good general combiners for cob girth and KRN traits. Similarly, PML 109, PML 110, PML 111, PML 114, and PML 116 with significant GCA effects of 0.38, 0.46, 0.41, 0.36 and 0.56 respectively, found good general combiner for grain yield.Among the testers used, PML 46 and PML 93 had non-significant GCA effect for KRN (-0.33 and − 0.12) and grain yield (0.12 and 0.12). However, PML 102, other inbred line used as tester, showed significant GCA effect for both KRN (0.45) and grain yield (-0.24) , AH-4316 (PML 110 × PML 46), AH-4304 (PML 111 × PML 46), AH-4305 (PML 109 × PML 93) and AH-4334 (PML 114 × PML 102) showed significantly superior grain yield over the medium maturing national check hybrid, Bio-9544 (Supplementary Table 6).Breeding for hybrid in any crop is one of the finest interventions of agriculture innovation which has directly impact on increasing in productivity. Understanding heterosis from the perspective of any single mechanisms alone may be elusive, because heterosis is likely an emergent property of populations . Hybrid Heterosis is the function of allelic diversity and degree of dominance of a trait harbor in the parental lines, which are exploited during the development of hybrids . AllelicThe potentiality of inbred lines favoring heterosis can be identified by their combining ability studies. In the present investigation, a set of 13 female (lines) and 3 male (tester) parental lines were identified and crossed into line x testers fashion and generated 39 test cross hybrids. The analysis of variance for combining ability suggested that there was significant variation due to cross or entries for all the traits studied, which in turn suggested the presence of wider genetic diversity among different traits. Furthermore, the partitioning of the mean sum of squares attribute to different sources of variation revealed that mean sum of squares due to lines and its crosses were highly significant. Also, there was significant variation due to lines and testers for all the traits under studied except KPR; hence, there is a high genetic divergence between lines and testers [The interaction between line and tester was showed significant differences for grain yield trait than the rest of traits. Therefore, testers used in the hybrid combinations were better differentiated for productivity, the contribution towards variance due to hybrids could be better accounted for grain yield. Hence, as advocated, this design gives better insights to the performance of the lines and testers involved in the series of cross combinations .The complete understanding of genetic basis of heterosis and combining ability remains elusive, which, however, does not affect the vital role of heterosis and combining ability in general, and maize breeding, in particular. Although there are still some gaps to understand the mechanism of heterosis, but great progress has been made in predicting hybrid performance based on the combining ability studies . In the Among the testers, PML 102 shows significant positive GCA effect for KRN (0.45) and negative GCA effect for yield (-0.24). Similarly, PML 93 and PML 46 showed non-significant positive GCA effect for KRN and yield . TherefoThe usefulness of a particular cross involving diverse parental lines in exploiting heterosis phenomenon is judged by the SCA effect of the component lines. According to Sprague and Tatum , SCA is per se performance of female inbred lines plays crucial role in economic seed production of any hybrids, breeder may tend to select even poor performing female parents, if genetic distance between the parents is high, will be explore for heterosis breeding programme [per se grain yield, the female inbred line PML 116 had contributed promising hybrids, as this line was having significant GCA with additive effect for KRN and grain yield. Hence, breeder should trade-off between per se performance of the lines and their genetic diversity after repeated evaluation.Although rogramme , 27. Therogramme –32. In tper se. Test cross performance gives some idea about breeding value of the lines under testing. The present investigation identified the wider genetic variability among inbred lines under study. Based on combining ability analysis (GCA and SCA), inbred line showed both additive and epistatic effect for yield and yield attribute traits and also showed distinct divergence in inbred lines based on molecular diversity approach.Understanding the breeding value of the parental lines in hybrid breeding program plays a paramount role in increasing hybrid yield Below is the link to the electronic supplementary material.Supplementary Material 1