Comparison of the abilities of ambient and manufactured nanoparticles to induce cellular toxicity according to an oxidative stress paradigm

Nanomaterial properties differ from those bulk materials of the same composition, allowing them to execute novel activities. A possible downside of these capabilities is harmful interactions with biological systems, with the potential to generate toxicity. An approach to assess the safety of nanomaterials is urgently required. We compared the cellular effects of ambient ultrafine particles with manufactured titanium dioxide (TiO2), carbon black, fullerol, and polystyrene (PS) nanoparticles (NPs). The study was conducted in a phagocytic cell line (RAW 264.7) that is representative of a lung target for NPs. Physicochemical characterization of the NPs showed a dramatic change in their state of aggregation, dispersibility, and charge during transfer from a buffered aqueous solution to cell culture medium. Particles differed with respect to cellular uptake, subcellular localization, and ability to catalyze the production of reactive oxygen species (ROS) under biotic and abiotic conditions. Spontaneous ROS production was compared by using an ROS quencher (furfuryl alcohol) as well as an NADPH peroxidase bioelectrode platform. Among the particles tested, ambient ultrafine particles (UFPs) and cationic PS nanospheres were capable of inducing cellular ROS production, GSH depletion, and toxic oxidative stress. This toxicity involves mitochondrial injury through increased calcium uptake and structural organellar damage. Although active under abiotic conditions, TiO2 and fullerol did not induce toxic oxidative stress. While increased TNF-alpha production could be seen to accompany UFP-induced oxidant injury, cationic PS nanospheres induced mitochondrial damage and cell death without inflammation. In summary, we demonstrate that ROS generation and oxidative stress are a valid test paradigm to compare NP toxicity. Although not all materials have electronic configurations or surface properties to allow spontaneous ROS generation, particle interactions with cellular components are capable of generating oxidative stress.     

Mechanistic Investigation of the Biological Effects of SiO2, TiO2, and ZnO Nanoparticles on Intestinal Cells

Silicon dioxide (SiO₂), titanium dioxide (TiO₂), and zinc oxide (ZnO) are currently among the most widely used nanoparticles (NPs) in the food industry. This could potentially lead to unintended exposure of the gastrointestinal tract to these NPs. This study aims to investigate the potential side‐effects of these food‐borne NPs on intestinal cells and to mechanistically understand the observed biological responses. Among the panel of tested NPs, ZnO NPs are the most toxic. Consistently in all three tested intestinal cell models, ZnO NPs invoke the most inflammatory responses from the cells and induce the highest intracellular production of reactive oxygen species (ROS). The elevated ROS levels induce significant damage to the DNA of the cells, resulting in cell‐cycle arrest and subsequently cell death. In contrast, both SiO₂ and TiO₂ NPs elicit minimum biological responses from the intestinal cells. Overall, the study showcases the varying capability of the food‐borne NPs to induce a cellular response in the intestinal cells. In addition to physicochemical differences in the NPs, the genetic landscape of the intestinal cell models governs the toxicology profile of these food‐borne NPs.     

Spatiotemporal Catalytic Nanozymes Microneedle Patches with Opposite Properties for Wound Management

Reactive oxygen species (ROS)-mediated biological catalysis involves serial programmed enzymatic reactions and plays an important part against infectious diseases; while the spatiotemporal control of catalytic treatment to break the limitations of the disease microenvironment is challenging. Here, a novel spatiotemporal catalytic microneedles patch (CMSP-MNs) integrated with dual-effective Cu₂MoS₄ (CMS) and polydopamine (PDA) nanoparticles (NPs) for breaking microenvironment restrictions to treat wound infections is designed. Since CMS NPs are loaded in the needles, CMSP-MNs can catalytically generate diverse ROS to cause effective bacterial inactivation during bacterial infection process. Besides, PDA NPs are encapsulated in the backing layer, which facilitate ROS elimination and oxygen production for solving hypoxic problems in wound microenvironment and alleviating the expression of inflammatory factors during the inflammation process. Based on these features, it is demonstrated through cell and animal experiments that these nanozymes-integrated MNs patches can realize selective regulation of ROS level with bacterial inactivation and inflammatory treatment, resulting in minimized side effects of over-production ROS and effective anti-infected treatment. It is believed that the presented MNs can provide a new therapeutic strategy with spatiotemporal adjustable catalytic properties in biomedical areas.     

A Mussel‐Inspired Persistent ROS‐Scavenging,Electroactive, and Osteoinductive Scaffold Based on Electrochemical‐Driven In Situ Nanoassembly

Conductive polymers are promising for bone regeneration because they can regulate cell behavior through electrical stimulation; moreover, they are antioxidative agents that can be used to protect cells and tissues from damage originating from reactive oxygen species (ROS). However, conductive polymers lack affinity to cells and osteoinductivity, which limits their application in tissue engineering. Herein, an electroactive, cell affinitive, persistent ROS‐scavenging, and osteoinductive porous Ti scaffold is prepared by the on‐surface in situ assembly of a polypyrrole‐polydopamine‐hydroxyapatite (PPy‐PDA‐HA) film through a layer‐by‐layer pulse electrodeposition (LBL‐PED) method. During LBL‐PED, the PPy‐PDA nanoparticles (NPs) and HA NPs are in situ synthesized and uniformly coated on a porous scaffold from inside to outside. PDA is entangled with and doped into PPy to enhance the ROS scavenging rate of the scaffold and realize repeatable, efficient ROS scavenging over a long period of time. HA and electrical stimulation synergistically promote osteogenic cell differentiation on PPy‐PDA‐HA films. Ultimately, the PPy‐PDA‐HA porous scaffold provides excellent bone regeneration through the synergistic effects of electroactivity, cell affinity, and antioxidative activity of the PPy‐PDA NPs and the osteoinductivity of HA NPs. This study provides a new strategy for functionalizing porous scaffolds that show great promise as implants for tissue regeneration.     

Mechanistic Differences in Cell Death Responses to Metal‐Based Engineered Nanomaterials in Kupffer Cells and Hepatocytes

The mononuclear phagocyte system in the liver is a frequent target for nanoparticles (NPs). A toxicological profiling of metal‐based NPs is performed in Kupffer cell (KC) and hepatocyte cell lines. Sixteen NPs are provided by the Nanomaterial Health Implications Research Consortium of the National Institute of Environmental Health Sciences to study the toxicological effects in KUP5 (KC) and Hepa 1–6 cells. Five NPs (Ag, CuO, ZnO, SiO₂, and V₂O₅) exhibit cytotoxicity in both cell types, while SiO₂ and V₂O₅ induce IL‐1β production in KC. Ag, CuO, and ZnO induced caspase 3 generated apoptosis in both cell types is accompanied by ion shedding and generation of mitochondrial reactive oxygen species (ROS) in both cell types. However, the cell death response to SiO₂ in KC differs by inducing pyroptosis as a result of potassium efflux, caspase 1 activation, NLRP3 inflammasome assembly, IL‐1β release, and cleavage of gasdermin‐D. This releases pore‐performing peptide fragments responsible for pyroptotic cell swelling. Interestingly, although V₂O₅ induces IL‐1β release and delays caspase 1 activation by vanadium ion interference in membrane Na⁺/K⁺ adenosine triphosphate (ATP)ase activity, the major cell death mechanism in KC (and Hepa 1–6) is caspase 3 mediated apoptosis. These findings improve the understanding of the mechanisms of metal‐based engineered nanomaterial (ENM) toxicity in liver cells toward comprehensive safety evaluation.     

Effect of the surface texture and crystallinity of ZnO nanoparticles on their toxicity

We have investigated the correlation between the structural properties of ZnO nanoparticles (NPs) and their toxicity to mesenchymal stem cells (C2C12 cell line) and macrophage-derived cells (RAW 264.7 cell line). Nanopowders of grain size ranging between 5 nm and 50 nm were prepared by chemical route. Their structural properties were characterized extensively by X-ray Diffraction (XRD) and High Resolution Transmission Electron Microscopy (HRTEM). The XRD spectra showed that 50 nm sized NPs are well crystallized and present a preferential orientation along the direction normal to the (001) plane while the HREM observations revealed that most of the large size (50 nm) crystallized nanoparticles have polygonal shape which is consistent with a texture of along [001] direction. The toxicity tests showed that [001] large textured NPs have higher toxicity to inflammatory cells than nanoparticles of low crystallinity and much smaller size (5 nm). In addition, NPs have cytotoxic effects on inflammatory cells at concentration as low as 0.05 mM while ten times higher concentrations did not have significant cytotoxic effects on cells representing mesenchymal tissues. These observations are explained by the enhanced generation of Reactive Oxygen Species (ROS) at the (0001) polar surface of ZnO NP. These results provide a direct evidence of the correlation between the toxicity and the surface texture of the oxide nanoparticles. Similar correlation has been reported for the photocatalytic properties of ZnO nanoparticles.     

HClO-Activated Fluorescence and Photosensitization from an AIE Nanoprobe for Image-Guided Bacterial Ablation in Phagocytes

Bacteria hiding in host phagocytes are difficult to kill, which can cause phagocyte disorders resulting in local and systemic tissue damage. Effective accumulation of activatable photosensitizers (PSs) in phagocytes to realize selective imaging and on-demand photodynamic ablation of bacteria is of great scientific and practical interests for precise bacteria diagnosis and treatment. Herein, HClO-activatable theranostic nanoprobes, DTF-FFP NPs, for image-guided bacterial ablation in phagocytes are introduced. DTF-FFP NPs are prepared by nanoprecipitation of an HClO-responsive near-infrared molecule FFP and an efficient PS DTF with aggregation-induced emission characteristic using an amphiphilic polymer Pluronic F127 as the encapsulation matrix. As an energy acceptor, FFP can quench both fluorescence and production of reactive oxygen species (ROS) of DTF, thus eliminating the phototoxicity of DTF-FFP NPs in normal cells and tissues. Once delivered to the infection sites, DTF-FFP NPs light up with red fluorescence and efficiently generate ROS owing to the degradation of FFP by the stimulated release of HClO in phagocytes. The selective activation of fluorescence and photosensitization is successfully confirmed by both in vitro and in vivo results, demonstrating the effectiveness and theranostic potential of DTF-FFP NPs in precise bacterial therapy.     

Interaction of silver nanoparticles with an environmentally beneficial bacterium, Pseudomonas chlororaphis

This study explores the potential antimicrobial mechanisms of commercial silver nanoparticles (Ag NPs) in the environmental bacterium, Pseudomonas chlororaphis O6. The 10nm size NPs aggregated in water, as demonstrated by atomic force microscopy. Solubility of the NPs at 10mg/L was 0.28 mg/L (pH 6) and 2.3mg/L (pH 7); release from 10mg/L bulk Ag was below detection. The NPs eliminated cell culturability at 3mg/L, whereas no effect was observed at 10mg/L bulk Ag. Zeta potential measurements revealed that the NPs were negatively charged; unlike Ag ions, their addition to the negatively charged cells did not change cell charge at pH 6, but showed a trend to reduce cell charge at pH 7. Isolated extracellular polymeric substances (EPS) from PcO6 was polydisperse, with negative charge that was neutralized by Ag ions, but not by the NPs. Addition of EPS eliminated Ag NP's toxicity in cells lacking EPS. Intracellular accumulation of OH was not detected in NP-treated cells; however, the use of scavengers suggested the NPs caused extracellular H(2)O(2) production. No evidence was found for loss of membrane integrity upon treatment with the NPs. Our findings indicate that growth of environmental bacteria could be impaired by Ag NPs, depending on the extent of EPS production.     

Penetration,distribution and brain toxicity of titanium nanoparticles in rodents' body: a review

Titanium dioxide (TiO₂) has been vastly used commercially, especially as white pigment in paints, colorants, plastics, coatings, cosmetics. Certain industrial uses TiO₂ in diameter <100 nm. There are three common exposure routes for TiO₂ : (i) inhalation exposure, (ii) exposure via gastrointestinal tract, (iii) dermal exposure. Inhalation and gastrointestinal exposure appear to be the most probable ways of exposure, although nanoparticle (NP) penetration is limited. However, the penetration rate may increase substantially when the tissue is impaired. When TiO₂ NPs migrate into the circulatory system, they can be distributed into all tissues including brain. In brain, TiO₂ lead to oxidative stress mediated by the microglia phagocytic cells which respond to TiO₂ NPs by the production and release of superoxide radicals that convert to multiple reactive oxygen species (ROS). The ROS production may also cause the damage of blood–brain barrier which then becomes more permeable for NPs. Moreover, several studies have showed neuron degradation and the impairment of spatial recognition memory and learning abilities in laboratory rodent exposed to TiO₂ NPs.Inspec keywords: nanoparticles, permeability, health hazards, molecular biophysics, biochemistry, biological tissues, toxicology, oxidation, brain, neurophysiology, blood, reviews, cosmetics, cellular biophysics, titanium compoundsOther keywords: rodents, white pigment, gastrointestinal tract, dermal exposure, gastrointestinal exposure, nanoparticle penetration, penetration rate, blood–brain barrier, titanium nanoparticles, brain toxicity, size 100.0 nm, TiO₂      

Mechanisms of Antibacterial Activity of MgO: Non‐ROS Mediated Toxicity of MgO Nanoparticles Towards Escherichia coli

The toxicity of metal oxide nanomaterials and their antimicrobial activity is attracting increasing attention. Among these materials, MgO is particularly interesting as a low cost, environmentally‐friendly material. The toxicity of MgO, similar to other metal oxide nanomaterials, is commonly attributed to the production of reactive oxygen species (ROS). We investigated the toxicity of three different MgO nanoparticle samples, and clearly demonstrated robust toxicity towards Escherichia coli bacterial cells in the absence of ROS production for two MgO nanoparticle samples. Proteomics data also clearly demonstrate the absence of oxidative stress and indicate that the primary mechanism of cell death is related to the cell membrane damage, which does not appear to be due to lipid peroxidation.     

Biomimicry 3D Gastrointestinal Spheroid Platform for the Assessment of Toxicity and Inflammatory Effects of Zinc Oxide Nanoparticles

Our current mechanistic understanding on the effects of engineered nanoparticles (NPs) on cellular physiology is derived mainly from 2D cell culture studies. However, conventional monolayer cell culture may not accurately model the mass transfer gradient that is expected in 3D tissue physiology and thus may lead to artifactual experimental conclusions. Herein, using a micropatterned agarose hydrogel platform, the effects of ZnO NPs (25 nm) on 3D colon cell spheroids of well‐defined sizes are examined. The findings show that cell dimensionality plays a critical role in governing the spatiotemporal cellular outcomes like inflammatory response and cytotoxicity in response to ZnO NPs treatment. More importantly, ZnO NPs can induce different modes of cell death in 2D and 3D cell culture systems. Interestingly, the outer few layers of cells in 3D model could only protect the inner core of cells for a limited time and periodically slough off from the spheroids surface. These findings suggest that toxicological conclusions made from 2D cell models might overestimate the toxicity of ZnO NPs. This 3D cell spheroid model can serve as a reproducible platform to better reflect the actual cell response to NPs and to study a more realistic mechanism of nanoparticle‐induced toxicity.     

Cytotoxicity of titanium dioxide nanoparticles differs in four liver cells from human and rat

Titanium dioxide (TiO₂) nanoparticles (NPs) are the important nanoscale components of composites. Although TiO₂ NPs and their related nanocomposites have been widely used in industrial and medical applications, the adverse effects of TiO₂ nanomaterials have not been well studied. Here, we investigated the cytotoxicity of TiO₂ NPs in vitro using four liver cell lines: human hepatocellular carcinoma cell line (SMMC-7721), human liver cell line (HL-7702), rat hepatocarcinoma cell line (CBRH-7919) and rat liver cell line (BRL-3A). We checked cell viability, cell morphology, and the levels of reactive oxygen species (ROS) and glutathione (GSH) after TiO₂ exposure at varying concentrations (0.1–100 μg/mL) and different exposure periods of time (12–48 h). Compared to the NP-free control, all four cell lines exposed to TiO₂ NPs showed cytotoxicity in a dosage-dependent and time-dependent manner, which was associated with the changes of cell viability and cell morphology, increased intercellular ROS levels, and decreased intracellular GSH levels. Further, we observed that carcinomatous liver cells and human liver cells exhibited more tolerance to TiO₂ NPs exposure for 24 h, compared to normal liver cells and rat liver cells, respectively. The results indicate that the in vitro cytotoxicity induced by NPs should be assessed with great caution before the use of nanocomposites and that there is a need to standardize the cytotoxicity testing procedure of nanoscale components in composites when using different cell lines.     

Self-Assembly of Selenium-Doped Carbon Quantum Dots as Antioxidants for Hepatic Ischemia-Reperfusion Injury Management

Hepatic ischemia-reperfusion injury (HIRI) is a critical complication after liver surgery that negatively affects surgical outcomes of patients with the end-stage liver-related disease. Reactive oxygen species (ROS) are responsible for the development of ischemia-reperfusion injury and eventually lead to hepatic dysfunction. Selenium-doped carbon quantum dots (Se-CQDs) with an excellent redox-responsive property can effectively scavenge ROS and protect cells from oxidation. However, the accumulation of Se-CQDs in the liver is extremely low. To address this concern, the fabrication of Se-CQDs-lecithin nanoparticles (Se-LEC NPs) is developed through self-assembly mainly driven by the noncovalent interactions. Lecithin acting as the self-assembly building block also makes a pivotal contribution to the therapeutic performance of Se-LEC NPs due to its capability to react with ROS. The fabricated Se-LEC NPs largely accumulate in the liver, effectively scavenge ROS and inhibit the release of inflammatory cytokines, thus exerting beneficial therapeutic efficacy on HIRI. This work may open a new avenue for the design of self-assembled Se-CQDs NPs for the treatment of HIRI and other ROS-related diseases.     

Peptide-Functionalized Prussian Blue Nanomaterial for Antioxidant Stress and NIR Photothermal Therapy against Alzheimer's Disease

Excessive accumulations of reactive oxygen species (ROS) and amyloid-β (Aβ) protein are closely associated with the complex pathogenesis of Alzheimer's disease (AD). Therefore, approaches that synergistically exert elimination of ROS and dissociation of Aβ fibrils are effective therapeutic strategies for correcting the AD microenvironment. Herein, a novel near infrared (NIR) responsive Prussian blue-based nanomaterial (PBK NPs) is established with excellent antioxidant activity and photothermal effect. PBK NPs possess similar activities to multiple antioxidant enzymes, including superoxide dismutase, peroxidase, and catalase, which can eliminate massive ROS and relieve oxidative stress. Under the NIR irradiation, PBK NPs can generate local heat to disaggregate Aβ fibrils efficiently. By modifying CKLVFFAED peptide, PBK NPs display obvious targeting ability for blood–brain barrier penetration and Aβ binding. Furthermore, in vivo studies demonstrate that PBK NPs have outstanding ability to decompose Aβ plaques and alleviate neuroinflammation in AD mouse model. Overall, PBK NPs provide evident neuroprotection by reducing ROS levels and regulating Aβ deposition, and may accelerate the development of multifunctional nanomaterials for delaying the progression of AD.     

ROS‐Responsive Polyprodrug Nanoparticles for Triggered Drug Delivery and Effective Cancer Therapy

The application of nanoparticles (NPs) to drug delivery has led to the development of novel nanotherapeutics for the treatment of various diseases including cancer. However, clinical use of NP‐mediated drug delivery has not always translated into improved survival of cancer patients, in part due to the suboptimal properties of NP platforms, such as premature drug leakage during preparation, storage, or blood circulation, lack of active targeting to tumor tissue and cells, and poor tissue penetration. Herein, an innovative reactive oxygen species (ROS)‐responsive polyprodrug is reported that can self‐assemble into stable NPs with high drug loading. This new NP platform is composed of the following key components: (i) polyprodrug inner core that can respond to ROS for triggered release of intact therapeutic molecules, (ii) polyethylene glycol (PEG) outer shell to prolong blood circulation; and (iii) surface‐encoded internalizing RGD (iRGD) to enhance tumor targeting and tissue penetration. These targeted ROS‐responsive polyprodrug NPs show significant inhibition of tumor cell growth both in vitro and in vivo.     

Tuning Cellular Response to Nanoparticles via Surface Chemistry and Aggregation

The aggregation of gold nanoparticles (Au NPs) in cell media is a common phenomenon that can influence NP‐cell interactions. Here, we control Au NP aggregation in cell media and study the impact of Au NP aggregation on human dermal fibroblast (HDF) cells. By first adding Au NPs to fetal bovine serum (FBS) and then subsequently to a buffer, aggregation can be avoided. Aggregation of Au NPs also can be avoided by coating Au NPs with other biomolecules such as lipids. The aggregation state of the Au NPs influences cellular toxicity and Au NP uptake: non‐aggregated cationic Au NPs are four‐fold less toxic to HDF cells than aggregated cationic Au NPs, and the uptake of non‐aggregated anionic citrate Au NPs is three orders of magnitude less than that of aggregated citrate Au NPs. Upon uptake of Au NPs, cellular F‐actin fiber formation is disrupted and actin dots are predominant. When lipid‐coated Au NPs are doped with a fluorescent lipid (F‐lipid) and incubated with HDF cells, the fluorescence from the F‐lipid was found throughout the cell, showing that lipids can dissociate from the Au NP surface upon entering the cell.     

Toxicity of copper oxide nanoparticles: a review study

Copper oxide nanoparticles (CuO NPs) use has exponentially increased in various applications (such as industrial catalyst, gas sensors, electronic materials, biomedicines, environmental remediation) due to their flexible properties, i.e. large surface area to volume ratio. These broad applications, however, have increased human exposure and thus the potential risk related to their short‐ and long‐term toxicity. Their release in environment has drawn considerable attention which has become an eminent area of research and development. To understand the toxicological impact of CuO NPs, this review summarises the in‐vitro and in‐vivo toxicity of CuO NPs subjected to species (bacterial, algae, fish, rats, human cell lines) used for toxicological hazard assessment. The key factors that influence the toxicity of CuO NPs such as particle shape, size, surface functionalisation, time–dose interaction and animal and cell models are elaborated. The literature evidences that the CuO NPs exposure to the living systems results in reactive oxygen species generation, oxidative stress, inflammation, cytotoxicity, genotoxicity and immunotoxicity. However, the physio‐chemical characteristics of CuO NPs, concentration, mode of exposure, animal model and assessment characteristics are the main perspectives that define toxicology of CuO NPs.Inspec keywords: catalysts, nanofabrication, reviews, oxidation, toxicology, gas sensors, cellular biophysics, copper compounds, nanoparticles, biochemistryOther keywords: copper oxide nanoparticles, environmental remediation, short‐ term toxicity, long‐term toxicity, human cell lines, CuO NPs exposure, physiochemical characteristics, mode of exposure, animal model, ssessment characteristics, toxicology, time‐dose interaction, oxidative stress, inflammation, cytotoxicity, genotoxicity, immunotoxicity, toxicological hazard assessment, algae species, bacterial species, fish, rats, CuO     

Cytotoxicity of BSA‐Stabilized Gold Nanoclusters: In Vitro and In Vivo Study

Gold nanoclusters (Au NCs) are one of the most promising fluorescent nanomaterials for bioimaging, targeting, and cancer therapy due to their tunable optical properties, yet their biocompatibility still remains unclear. Herein, the cytotoxicity of bovine serum albumin (BSA)‐stabilized Au NCs is studied by using three tumor cell lines and two normal cell lines. The results indicate that Au NCs induce the decline of cell viabilities of different cell lines to varying degrees in a dose‐ and time‐dependent manner, and umbilical vein endothelial cells which had a higher intake of Au NCs than melanoma cells show more toxicity. Addition of free BSA to BSA‐Au NCs solutions can relieve the cytotoxicity, implying that BSA can prevent cell damage. Moreover, Au NCs increase intracellular reactive oxygen species (ROS) production, further causing cell apoptosis. Furthermore, N‐acetylcysteine, a ROS scavenger, partially reverses Au NCs‐induced cell apoptosis and cytotoxicity, indicating that ROS might be one of the primary reasons for the toxicity of BSA‐Au NCs. Surprisingly, Au NCs with concentrations of 5 and 20 nM significantly inhibit tumor growth in the xenograft mice model of human liver cancer, which might provide a new avenue for the design of anti‐cancer drug delivery vehicles.     

One‐pot synthesis chlorin e6 nano‐precipitation for colorectal cancer treatment Ce6 NPs for colorectal cancer treatment

The drug nanoparticles free of additional carriers hold great promise in drug delivery and are suitable for the therapy of cancers. Herein, we developed a one‐pot method to prepare chlorin e6 (Ce6) nano‐precipitations (Ce6 NPs) for effective photodynamic therapy of colorectal cancer. The drug loading of Ce6 NPs was around 81% and showed acceptable stability, high biocompatibility as well as effective reactive oxygen species (ROS) generation capability. As a result, the Ce6 NPs can produce significantly elevated ROS upon laser irradiations and achieved better anticancer benefits than free Ce6.