Currently, there is no efficient therapy for spinal cord injury (SCI). Anoxemia after SCI is a key problem, which leads to tissue destruction, while hypoxia after SCI induces cell injury along with inflammation. Mixed-lineage kinase domain-like protein (MLKL) is a critical signal molecule of necroptosis, and mitochondrial dysfunction is regarded as one of the most pivotal events after SCI. Based on the important role of MLKL in cell damage and potential role of mitochondrial dysfunction, our study focuses on the regulation of MLKL by Necrosulfonamide (NSA) in mitochondrial dysfunction of oxygen-glucose deprivation (OGD)-induced cell damage and SCI-mice, which specifically blocks the MLKL. Our results showed that NSA protected against a decrease in the mitochondrial membrane potential, adenosine triphosphate, glutathione, and superoxide dismutase levels and an increase in reactive oxygen species and malonyldialdehyde levels. NSA also improved the locomotor function in SCI-mice and OGD-induced spinal neuron injury through inhibition of MLKL activation independently of receptor-interacting protein kinase 3 (RIP3) phosphorylation. Besides the protective effects, NSA exhibited a therapeutic window. The optimal treatment time was within 12 h after the injury in the SCI-mice model. In conclusion, our data suggest a close association between the NSA level inhibiting p-MLKL independently of RIP3 phosphorylation and induction of neurological impairment by improving antioxidative capacity after SCI. NSA ameliorates neurological impairment in SCI through inhibiting MLKL-dependent necroptosis. It also provides a theoretical basis for further research and application of NSA in the treatment of SCI. Spinal cord injury (SCI) represents a serious trauma to the central nervous system (CNS) and often causes a devastating impact on the motor function and many essential physiological functions . The maiSCI includes primary and secondary damage . The privia phosphorylation of the mitochondrial protein MLKL, thereby causing mitochondrial dysfunction. As a new mechanism for necrosis, necroptosis and mitochondrial structural and functional damage have gained considerable attention (Current research suggests that neuron death after SCI mainly involves necrosis and apoptosis . Necroptttention .Mitochondria are organelles that produce adenosine triphosphate (ATP) in mammalian cells. In addition to energizing cells, mitochondria also regulate the cell cycle, growth, differentiation, and apoptosis. There is cumulating evidence that mitochondrial dysfunction plays an important role in the progression of CNS diseases such as Parkinson’s disease, Alzheimer’s disease, cerebral ischemic stroke, Huntington disease, multiple sclerosis, and amyotrophic lateral sclerosis . Furtherin vitro through RIP3 and MLKL activation , which specifically blocks the MLKL, for preventing mitochondrial dysfunction after SCI. It has been shown that NSA impedes SCI by inhibiting necroptosis . Zhou ettivation . We alsoC57BL/6 mice  were anesthetized through inhalation of 3% isoflurane with a flow rate of 1 L/min. The SCI model is described previously . BrieflyChatillon  was used for measuring forelimb grip strength in order to evaluate the SCI. As described in our previous report , mice weThe Basso Mouse Scale (BMS) measured the hind limb movements during locomotion in an open field through a 0- to 9-point scale (a high score means the better hind limb function). This assessment was performed independently by two blinded researchers over 4 min at pre-injury and 1, 2, 3, 7, 14, 21, and 28 days after SCI (n  =  9 mice/group).Spinal cord contusion was evaluated by calculating the water content of the spinal cord. The damaged spinal cord was dried at 100°C for 24 h, and the spinal cord water content was calculated using the following formula: [(wet weight—dry weight)/wet weight] × 100.3-pieces, after gently peeling off the meninges. The tissue pieces were digested by 0.25% trypsin for 10 min at 37°C. The cells were re-suspended in a serum-free medium, including Neurobasal, B27, and penicillin/streptomycin (50 U/ml), adjusted at a density of 1–5×105 cell/ml and seeded in 24-well plate at 37°C with 5% CO2. The spinal cord neurons were cultured for 10 days and then subjected to OGD stimulation. Briefly, the cells were incubated in Earle’s balanced salt solution  and cultured in an incubator containing 95% N2/5% CO2 for 4 h. Then, the EBSS was replaced with standard neuronal culture medium for 24 h. Cells cultured in standard neuronal culture medium in the presence of ambient 5% CO2 served as controls. After OGD treatment, neurons were immediately treated with either NSA  or Nec-1 . The vehicle group received an equal volume of DMSO.Three-day-old C57BL/6 neonatal pups were anesthetized by isoflurane in the airtight boxes and decapitated and had their vertebral columns removed. The spinal cord was isolated and cut into 1 mm® AQueous One Solution Cell Proliferation Assay . Cells were plated in 96-well plates in triplicate at approximately 3×104 cells per well and cultured in the growth medium. After treatment, MTS was added to the culture medium and incubated for 2 h at 37°C in humidified 95% air and 5% CO2. The absorbance was measured at 450 nm using a microplate reader .Cell damage was measured using the CellTiter 96ATP was detected through an ATP Content Assay Kit  following the manufacturer’s instructions. Cells were ultrasonically disrupted and centrifuged at 800 × g for 10 min at 4°C. Creatine kinase catalyses the reaction between creatine and ATP to produce phosphocreatine. To detect the ATP level, the content of phosphocreatine was determined by the colorimetric method of phosphomolybdic acid. The absorbance at 700 nm was measured by a microplate reader (Thermo Scientific).MMP was detected with JC-1 (Solarbio); 20 µl of JC-1 solution (0.15 mM in DMSO) was added to 100 μl of each suspension and was incubated at 37°C for 20 min; then, JC-1 monomer was detected at 490 nm excitation and 530 nm emission wavelengths, and JC-1 polymer was detected at 5,250 nm excitation and 590 nm emission wavelengths using a fluorescence spectrophotometer (Thermo Scientific).in vitro were collected and lysed; then, 100 µl of the supernatant, 100 µl of oxidized glutathione solution, and 20 µl of NADPH solution (6 mM) were mixed, and GSH was detected in the supernatant at 405 nm.The activities of SOD, MDA, and GSH were detected according to the manufacturer’s protocol . Homogenate was obtained from tissue or cell, centrifuged for 10 min at 10,000 × g at 4°C, and the supernatant was stored at −80°C. For SOD detection, the reaction system contained 20 µl WST-1 enzyme reaction solution, 20 µl sample, and 200 µl enzyme substrate. The reaction mixtures were incubated at 37°C for 20 min. One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of the reduction of WST-1 formazan detected at 450 nm. For MDA detection, 20 µl of the sample and 200 µl of thiobarbituric acid reaction solution were mixed and heated at 95°C for 40 min. After cooling, the supernatant was obtained by centrifugation at 4,000 rpm for 10 min, and MDA concentration was detected in the supernatant at 532 nm. For GSH detection, erythrocytes from mouse whole blood or cells treated The ROS detection was performed according to the manufacturer’s instructions (Nanjing Jiancheng Bioengineering Institute). Following the indicated treatment, the monoplast suspensions were harvested and then, resuspended in 10 μM 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) solution and incubated in a CO2 incubator at 37°C for 30 min in the dark, followed by washing with PBS for three times. Finally, the cells were resuspended in 0.5 ml of PBS, and the fluorescence intensity of the samples was analysed using a fluorescence microplate.The total RNA was extracted from samples using TRIzol reagent (Solarbio). Total RNA was reversely transcribed to cDNA using a HiFi-MMLV cDNA First Strand Synthesis Kit . Quantitative real-time PCR was performed using GoTaq qPCR Master Mix (Promega) on the CFX96TM Real-Time System . Primer sequences were as follows: MLKL: F: 5´ -ACCCTTCAGAGGCACAACAC- 3´, R: 5´ -TGTCATTGGATTCGGTGGGG-3´; GAPDH: F: 5´ -AACTTTGGCATTGTGGAAGG- 3´, R: 5´ -GGATGCAGGGATGATGTTCT- 3´.Protein was harvested in 1×lysis buffer including a protease inhibitor. 30 µg total protein was separated by 10% SDS-PAGE, transferred to PVDF membrane, blocked for 1 h with TBST containing 5% BSA, and incubated at 4 °C overnight with the appropriate primary antibodies: RIP3 , pRIP3 , MLKL , p-MLKL , Bax , Bcl-2 , and GAPDH . They were then incubated with HRP-conjugated secondary antibodies , which was followed by chemiluminescent substrate development (Bio-Rad).Cells were fixed with 4% paraformaldehyde for 15 min at 37°C, washed three times with PBS, and then, incubated overnight at 4°C in PBS containing 2% Goat serum, 0.03% Triton X-100 in, p-MLKL antibody , and NeuN antibody . They were then incubated for 2 h at 25°C with the following fluorescent secondary antibodies. Cell nuclei were stained with DAPI, and images were obtained using a fluorescence microscope .Spinal cord neurons were harvested, and the damage was determined by DeadEnd™ Fluorometric Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) System (Promega). TUNEL staining was conducted with fluorescein-dUTP for apoptotic cell nuclei and DAPI stain for all cell nuclei. Images were obtained using a fluorescence microscope (Olympus).t-test or a one-way ANOVA, followed by Tukey’s multiple comparison test, with p < 0.05 considered statistically significant. Each experiment consisted of at least four replicates per condition. All data are described as the mean ± SEM.All statistical analyses were performed with SPSS 22.0 and analysed using the Student’s NSA specifically blocks MLKL, while Nec-1 is an inhibitor of RIP1. Therefore, they act as inhibitors of necroptosis. In the OGD-induced assay, the viability of NSA-treated cells at 3 μM and 10 μM (p < 0.05); however, NSA did not reduce OGD-induced high levels of p-RIP3 (Western Blot (WB) analysis and immunoﬂuorescence analyses were performed to assess the role of MLKL activity (p-MLKL level) in OGD-induced damage. NeuN and p-MLKL double staining was conducted in order to observe the effect of NSA on MLKL activation in spinal cord neurons. The results revealed that OGD at 4 h induced p-MLKL, while NSA relieved MLKL activation but not RIP3 activation . In NSA-treated groups , p-MLKL expression was reduced after SCI; however, it was still high in the NSA-treated group at 24 h . Redox status measurements were conducted in each group. SCI led to an increase in ROS and MDA levels and a decrease in SOD and GSH levels compared to the sham group . NSA treatment  resulted in a significant reduction in ROS and MDA levels and an increase in SOD and GSH levels compared to the SCI group . The effect on MMP and SOD was weaker in the 24 h group than that in the other NSA groups, but the 24 h group still revealed significant improvement in the ATP, ROS, MDA, and GSH levels compared to the SCI + vehicle group . Therefore, NSA demonstrated a more superior effect on mitochondrial dysfunction, including improvements in mitochondrial integrity and antioxidant capacity, within 12 h after SCI compared to the treatment at 24 h.The levels of p-RIP3 and p-MLKL were measured at 3 days after SCI (p < 0.0001). The data indicated that NSA protected against neuronal damage and promoted recovery post-SCI.As shown in in vitro  and the Specific Talent Project for Medical and Health of Jilin Province (2019SCZT031) supported this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Herein, we hypothesized that omega-3 fatty acids from flaxseed oil enriched diet disrupt the pro-inflammatory status in the retina, protecting against retinopathy development. For eight weeks under a high-fat diet (HF), several physiological parameters were monitored to follow the metabolic homeostasis disruption. After this period, mice were treated with a HF substituted in part of lard by flaxseed oil (FS) for another eight weeks. Food behavior, weight gain, glucose and insulin sensitivity, electroretinography, RT-qPCR and western blots were carried out. The HF was able to induce a pro-inflammatory background in the retina, changing IL1β and TNFα. VEGF, a master piece of retinopathy, had early onset increased also induced by HF. The FS-diet was able to decrease inflammation and retinopathy and improved retinal electro stimuli compared to HF group. GPR120 and GPR40 (G Protein-Coupled Receptors 120 and 40), an omega-3 fatty acid receptors, were detected in the retina for the first time. FS-diet modulated the gene expression and protein content of these receptors. Thus, unsaturated fatty acids protect the retina from diabetes type 2 mice model from disease progression.The chronic and low-grade inflammation induced by obesity seem to be the “ Once the fat accumulation-induced pro-inflammatory status induces a systemic glucose homeostasis dysfunction, the glucose uptake by skeletal muscle is decreased while gluconeogenesis by the liver is increased4. Retinal tissue is one of the leading susceptible tissue to acute hyperglycemia because the glucose transporter 1 (GLUT1), the primary glucose transporter in this tissue, is insulin independent and control by glucose active transport5.There is a global diabetes epidemic6, saturated fatty acids from diet7, or lipolysis of adipose tissue (free fatty acids)9 through Toll-Like Receptor (TLR) and interleukin 1 receptor (IL1R) pathways. VEGF  is considered the main marker involved in retinal damage, once the enhance of intraocular inflammatory process11 by the increase of vessel-permeability13 results in pathological angiogenesis, which is irregularly distributed with poorly constructed vessels prone to leak, leading to fluid build-up within the retina14. In the long term, these changes are capable of leading to apoptosis of retinal neurons15. The VEGF inhibitors such as aflibercept and ranibizumab have demonstrated enormous benefits to patients with diabetic retinopathy16. However, these drugs show a high financial cost17 and refractoriness18. Therefore, different strategies need to be explored to control or postpone the disease progression.In the retina, the increased expression of pro-inflammatory proteins and growth factors could be induced by high glucose levels19 or vegetable oils [Alpha-linolenic acid ALA (C18:3)]20 have their anti-inflammatory properties extensively investigated on the whole body21, including in the retina23. In contrast, the deficient intake of ω3 contributes to retinal degeneration, once ω3 is an essential fatty acid in mammals and retinal composition24. Thus, some clinical trials evaluated this hypothesis. The AREDS2 study enrolled 4203 elderly participants at high risk of progression to advanced age-related macular degeneration, which received 1 g of ω3 for five years. In the primary analysis, there was no reduction of disease progression25. On the other hand, the PREDIMED study enrolled 3482 participants receiving at least 500 mg of dietary ω3 for six years. In this approach, the authors found a decreased risk of sight-threatening in diabetic retinopathy26.Omega-3 (ω3) fatty acids, which is obtained from marine sources [Eicosapentaenoic acid EPA (C20:5) and Docosahexaenoic acid DHA (C22:6)]28. The ω3-sensitized GPR120 activate and recruit the first downstream mediator (β-arrestin2 protein), which concomitantly recruits TAB1/2 from TLR2/4 and TNF-α pathways, disassembling these inflammatory cascades23 by TAK1 dephosphorylation, a nodal protein. Our research group has investigated this mechanism in several tissues such as liver, skeletal muscle, adipose27, aorta29, and hypothalamus28; however, under this point of view, the retina remains completely unexplored.In the present study, we evaluated the recently described G-Protein Coupled Receptor 120 (GPR120), the main receptor of ω3, which mediates its potent anti-inflammatory and insulin-sensitizing receptor responsesThe comprehension of molecular mechanisms modulated by ω3 fatty acids in the retinal tissue under hyperglycemic and pro-inflammatory state could contribute to increasing the knowledge regarding non-pharmacological treatment approaches.To test the ability of ω3 fatty acids, presented in its natural food matrix (flaxseed oil), to cross the blood-retinal barrier and activate its receptor (GPR120), a time course test was performed. Lean Swiss mice were gavaged with 500 μL of flaxseed oil and the retina was removed at different times for immunoprecipitation. We observed ω3 was able to activate GPR120 receptor through the interaction with β-arrestin2 protein after 180 minutes post-gavage Fig. . This apFour weeks under HF-diet was enough to increase the body weight in comparison with the CT group (P < 0.05) Fig. . At the To ensure that ω3-alpha-linolenic fatty acid C18:3) achieved efficiently the bloodstream after treatments with FS diet, we carried out the blood fatty acid profile  intake was increased on HF group when compared to the CT Fig. , howeverTo determine whether the exposure for 16 weeks to experimental diets was able to change the retinal function, an electroretinogram (ERG) was performed. After exposition to different luminous intensities, even under high body weight and glucose levels, HF group did not show differences among amplitudes of wave A, B or C Fig. , which aTo evaluate the presence and distribution in the retinal tissue of ω3 fatty acids receptors, we carried out three different approaches such as mRNA quantification by RT-qPCR, protein content by Western blot, and immunohistochemical photomicrography. The GPR120 and GPR40 were found in the retina of mice under all treatments Fig. ; howeverAfter 16 weeks of CT, HF- or FS-diets consumption, the pro- and anti-inflammatory markers were evaluated in the retina. The HF-diet induced inflammatory status, increasing significantly TNFα and IL1β gene expressions (P < 0.05) when compared to the CT group Fig. . This beInterestingly, 16 weeks under HF-diet were enough to increase the primary retinopathy marker, VEGF (P < 0.05) when compared to the CT group Fig. . Alson, 24. We tested an oral treatment protocols, with a rich and natural ω3 source (flaxseed oil) from diet, mimicking a real and rational possibility for being used by human beings, once fish oils today, unfortunately, could be contaminated by heavy metals (e.g. mercury)30.The chronic and low-grade inflammation processes contribute to the development of the early stages of diabetic retinopathy. Several studies have implicated ω3 fatty acids in the improvement of physiological and molecular characteristics in the retinal tissue. However, these investigations did not define the action mechanisms of the ω3 fatty acids21, aorta29, hypothalamus28, and renal cells27. Again, this is the first approach adopted to the retina. Connor et al.22 and Tikhonenko et al.23 treated mice with ω3 enriched diet and observed the increase in ω3 fatty acid profile content in the retina, but without functional tests for GPR120/40 receptors.Then, the primary aim of the present study was to determine the presence and functionality of ω3 receptors, GPR120 and GPR40, in the retina on the basal state. The GPR120 and GPR40 mRNAs Fig.  and prot31, able to mimic obesity comorbidities highly similar to humans32. Herein, compared to the CT-group, mice under HF-diet exposure during 8 weeks increased body mass and fasting blood glucose with consequently insulin resistance  bioconversion in the HF group when compared to FS or CT groups (P < 0.05). It could, adjacently to saturated fatty acids on HF-diet, to increase the pro-inflammatory status observed in the retina. Interestingly, the alpha linolenic fatty acids from FS group was partially bioconverted in other ω3 fatty acids longer ones (EPA and DHA), decreasing inflammation, such as suggested by Tikhonenko23 and Johansson34 groups; and reinforced by this work.Before to test the ω3 anti-inflammatory hypothesis in the retinal tissue, we induced mice to the obesity and hyperglycemic state through an HF-diet Fig. . Swiss mnce Fig. . After i27 and humans beings36 with obesity and its associated comorbidities. However, Baranowski et al. treated mice with dietary flaxseed oil and showed a reduction in the adipocyte size, alterations in several pro-inflammatory molecular parameters even without significant changes in the body weight20. These data are exciting once body weight loss could influence several biological parameters. This study  reinforces the current findings regarding the positive effects of “ω3 molecule” in inducing, per se, biological alterations independently of body weight loss. Again, this statement is emphasized once the FS- and HF-diets were caloric equivalently in this study.Then, the FS diet was able to reduce fasting blood glucose and glucose intolerance, improving whole body glucose homeostasis Fig.  even wit37. In fact, a reduction in the oscillatory potential was evidenced in mice treated with HF-diet, but not in FS, suggesting the compromised visual acuity  explored the increased expression and activity of TLR2 and TLR4 in type 2 diabetic subjects6. Additionally, Anan et al., (2010) and Tang & Kern (2011) found an association between retinopathy and visceral fat accumulation, attributing the retinopathy to pro-inflammatory state also in humans37.Hyperglycemia is a determinant factor capable of inducing alterations in the retina structure and, consequently, in the visual acuityity Fig. . Here, t14. In general, scientific literature reports VEGF as a late disease marker, which is firstly dependent on biochemical and hemodynamic alterations. The retinal endothelial cells have a high significantly amount of VEGF receptors, and the hypoperfusion and hypoxia induce VEGF expression. Therefore, VEGF is considered as a master angiogenesis regulator and a pro-inflammatory protein, leading to permeability by rapid tight-junctions’ (occludin and zonula occluden1) phosphorylation and disable13. The FS-diet was able to protect retina against VEGF raises.The HF-diet induced a pronounced pro-inflammatory state in the retinal tissue, changing the gene expression and protein content of the main pro-inflammatory markers (TNFα and IL1β), and activating the IL10, a homeostatic anti-inflammatory protein Fig. . On the 38. However, NF-κB is strongly regulated by a nodal TAK1 protein cascade, which is upstream activated by juxtamembrane-adjacent TLR, TNFα, and interleukin (IL1β and IL6) receptor proteins39. Herein, the ω3 in the flaxseed diet activated GPR120, which recruited β-arrestin2 and reduced TAK1 phosphorylation, disassembling the pro-inflammatory cascade triggered by high saturated fatty acids in the HF-diet.Also, VEGF gene is also controlled by NF-κB40; b) The normal ONL contains rod and cone photoreceptor nuclei throughout the retina41; c) INL consist of bipolar cells, which are directly synapsed to retinal ganglion cells and Müller glial cells42; d) and OPL has a certain degree of integration of the visual message takes place at the first synapse in the retina43.For the first time, both GPR120 and 40 were, through different approaches, identified, quantified and mapped in the retinal tissue Figs , while G44, monocyte chemotactic protein (MCP)-1, IL8, IL6, and interferon (IFN)-γ with or without stimulation by other pro-inflammatory cytokines45. However, overexpression of cytokines can impair its special features, causing retinal degeneration and even blindness40. Once controlling the pro-inflammatory cytokines background, GPR120 expressed in this layer could contribute to RPE function, maintaining ONL and INL layers integrated.Interestingly, in the basal state, RPE cells are involved in the production of VEGF43. The ONL and INL thickness provide an indirect measure of photoreceptor survival41; however, INL directly synapses to retinal ganglion cells and Müller glial cells, which are involved in retinal environment homeostasis. Retinal tissue is structured similarly to the central nervous system (CNS) and, for example, in the hypothalamus, GPR120 is expressed and recognizes ω3 fatty acids, disassembling inflammatory tonus induced by obesity state or triggered directly by saturated fatty acids28. As well in the CNS, Müller glial cells are throughout the main retinal layers and could also be responsive to fatty acids from the diet, controlling the local immune response and, consecutively, structural tissue integrity. The GPR120 receptor was also described in the liver, in the surface of Kuppfer cells, and in macrophages of the aorta endothelium, decreasing pro-inflammatory cytokines, reverting hepatic steatosis21 and atherosclerosis29, respectively, when mice were treated with flaxseed oil.OPL has integrative neurons and forms synapses to the bipolar, amacrine, and Müller glial cells in INL, which is closely integrated to ONL46. This percentage allows to GPR40 to be responsiveness for ω3 fatty acids21; however, without biological impact for this model. GPR40 is highly responsiveness to oleic fatty acid (ω9), and its role investigation remains an interesting issue.Despite the presence of GPR40 be present in retinal tissue Fig. , this reAltogether, these results describes an essential part of the role of GPR120, a central ω3 fatty acids receptor, such as summarized in the schematic Fig. ad libitum. The number of mice used in each experiment is detailed in subtitles of graphs.Swiss male mice (4 week-old) were provided by the University of Campinas Animal Breeding Center (CEMIB). All animal procedures were performed in accordance with guidelines and regulations, and approved by University of Campinas Ethical Committee (protocol #4197-1). The animals were housed in individual cages under 12 h dark/12h-light cycle, room temperature at 21 ± 2 °C, and water and diet Firstly, we tested whether there are GPR120 in the retina tissue and its activation time. Then, ω3 fatty acids from flaxseed oil was administered to mice (4 week-old), after 8 hours of fasting. The retina was removed immediately after administration (group 0 min) or after 120, 180, 210 or 240 min. GPR120 from retina was submitted to immunoprecipitation  with β-arrestin2 Fig. .®) or HF group, fed with a high-fat diet , for eight weeks. The high-fat diet was prepared in according to the American Institute of Nutrition (AIN-93G) guidelines47, modified to contain 35% of fat28  fatty acid content reached 52.3%. Therefore, the diets were prepared every seven days and maintained safely of light exposure at 4 °C. These groups were maintained in this protocol for another eight weeks. The control group was maintained in chow diet (CT)  Fig. .Table 2T−1 of insulin (Humulin R® - Lilly Indianapolis USA) was injected intraperitoneally (i.p.). Blood samples were collected at 0, 5, 10, 15, 20, 25 and 30 min from the tail vein for serum glucose determination in the electronic glucometer (Optium Xceed® - Abbott). The constant for the rate of serum glucose disappearance was calculated using the formula 0.693/biological half-life (t ½). The plasma glucose t ½ was calculated from the slope of the last square analysis of plasma glucose concentration during the linear phase of decline, as previously described48.These tests were carried out after eight hours of fasting and in two moments: 1) at the end of the obesity period induction, and 2) at the end of the experimental period. For ip.ITT, 1,5U/kg body weightFor ip.GTT, after 8 hours of fasting, a glucose solution (2 g/Kg body weight) was administered i.p. Blood samples were collected at 0, 30, 60, 90 and 120 min from the tail vein for serum glucose determination. Results were presented as the area under the curve.−1, respectively) under dim red illumination (λmax = 650 nm). Retinal function was measured using the UTAS-E3000 system  as previously described49. The pupils were dilated with tropicamide . The measurements were taken after dark overnight adaptation. An intensity-response series was recorded using a set of ganzfeld flashes with 0.25 cd.s/m2 intensity. Recordings were amplified and digitized using a 24-bit A/D converter band passed from 0.3 to 300 Hz with a 50-Hz notch filter.At the end of the experimental period, animals were submitted to full flash electroretinography. Thus, the animals were previously anesthetized with ketamine and xylazine  and xylazine (20 mg/Kg). The anesthesia was assured by the loss of corneal reflexes. After this, the eyeballs were removed. With the aid of a magnifying glass  and ophthalmic scalpel, an incision was made between the iris and internal ocular structures such as vitreous and crystalline. After this, the vitreous and crystalline were removed. Subsequently, with the aid of a forceps, the retina was removed, snap-frozen in liquid nitrogen, and stored at −80 °C until further analysis. In the end, mice were euthanized with anesthetics deepening.®, Connecticut, USA). The lysates were centrifuged at 12.000 rpm and 4 °C  for 40 minutes to remove the insoluble material. The supernatants were used for protein quantification, using the Bradford method50 and 40–60 μg were used for immunoblotting analysis. The samples were incubated at 95 °C for 5 minutes with Laemmili buffer. After this, samples were applied on a polyacrylamide gel for separation by SDS-polyacrylamide gel electrophoresis. Chemicals and buffers were from Bio-Rad . The proteins were transferred to nitrocellulose membranes. Binding of the antibody to nonspecific proteins was minimized by pre-incubation of the nitrocellulose membrane in 5% dry milk for 1 hour and then blotted with specific primary antibodies. Specific bands were labeled by chemiluminescence, visualization was performed with a system for chemiluminescence (G:BOX Chemi XRQ Syngene USA) and quantified using the software UN-SCAN-IT gel 6.1. The antibodies anti-βarrestin2 (sc13140), anti-GPR120 (sc48203), anti-GPR40 (sc32905), anti-IL-10 (sc1783), anti-TAK1 (sc7967), anti-VEGF (sc7269), anti-βactin (sc47778), and anti-GAPDH (sc25778) were purchased from Santa Cruz Biotechnologies. Antibody anti-Phospho-TAK1 [Ser412] (#9339) was purchased from Cell Signaling (Danvers MA USA). Antibodies anti-IL-1β (503502) and anti-TNF-α (506102) were purchased from BioLegend .The retinas were homogenized in extraction buffer . Precipitates were then analyzed by a Western blot with anti-GPR120 and re-probed with mouse anti-β-arrestin2.® reagent (LifeTechnologies Carlsbad CA USA). After this, the total RNA was isolated and quantified  and five microgram of total RNA from each sample were used to perform the digestion of genomic DNA (Recombinant DNase I RNase-free Takara). The cDNA reverse-transcription was performed using 1 µg of total RNA from retinal samples according to the instructions of the kit (High Capacity cDNA Reverse Transcription - Applied Biosystems). The 10 μL reaction mix consisted of 0.25 μL of primers, 3 µL of PCR master mix, 5 μL of template and DEPC water to achieve the final volume. Next, real-time PCR analysis of gene expression was performed in the StepOne Real-Time PCR Systems (Applied Biosystems). FFAR4/GPR120 (Mm00725193_m1), FFAR1/GPR40 (Mm00809442_m1), IL1β (Mm00434228_m1), TNFα (Mm00443258_m1), and GAPDH (Mm99999915_g1) were obtained from ThermoFisher Scientific.Total RNA was extracted from retinal tissue using TRIzolThe eyeballs were embedded in OCT compound  and sections (20 µm) were taken for the analysis. Sections were incubated in blocking solution (1 x PBS with 5% goat serum) followed by 2 hours of incubation at room temperature with primary antibodies against GPR120 (1:50) or GPR40 (1:10). After that, secondary antibodies conjugated to Alexa Fluor  were applied for 1 hour at room temperature. After PBS washes the sections were mounted with DAPI  for nucleic acid staining in blue. The eyes sections were examined under a fluorescence microscope . Digital images were captured using specific software .51. The fatty acids methyl esters were analysed with a gas chromatograph-mass spectrometer (Shimadzu® GCMS-QP2010 Ultra), and a fused-silica capillary Stabilwax column  with dimensions of 30 m × 0.25 mm internal diameter coated with a 0.25-µm thick layer of polyethylene glycol. The running conditions was followed in accordance with previous study52. Sample volumes of 1 µL were injected at 250 °C using a 20:1split ratio. High-grade pure helium (He) was used as the carrier gas with a constant flow rate of 1.0 mL/min. Mass conditions were as follows: ionization voltage, 70 eV; ion source temperature, 200 °C; full scan mode in the 35–500 mass range with 0.2 s/scan velocity.Lipids from blood were extracted following the proposed by Shirai (2005)t-test. After KS test, to compare CT, HF, and FS groups, one-way ANOVA was carried out and when significant (P < 0.05), Bonferroni’s test was used to compare the groups. All results were presented as the mean ± standard deviation with GraphPad Prism®, v5.0. software28.After normality test (Kolmogorov-Smirnov (KS)) the difference between CT and HF groups was considered higher than 5% (P < 0.05) using Student’s Supplementary Information


Protein Arginine (R) methylation is the most common post-translational methylation in mammalian cells. Protein Arginine Methyltransferases (PRMT) 1 and 5 dimethylate their substrates on R residues, asymmetrically and symmetrically, respectively. They are ubiquitously expressed and play fundamental roles in tumour malignancies, including glioblastoma multiforme (GBM) which presents largely deregulated Myc activity. Previously, we demonstrated that PRMT5 associates with Myc in GBM cells, modulating, at least in part, its transcriptional properties. Here we show that Myc/PRMT5 protein complex includes PRMT1, in both HEK293T and glioblastoma stem cells (GSCs). We demonstrate that Myc is both asymmetrically and symmetrically dimethylated by PRMT1 and PRMT5, respectively, and that these modifications differentially regulate its stability. Moreover, we show that the ratio between symmetrically and asymmetrically dimethylated Myc changes in GSCs grown in stem versus differentiating conditions. Finally, both PRMT1 and PRMT5 activity modulate Myc binding at its specific target promoters. To our knowledge, this is the first work reporting R asymmetrical and symmetrical dimethylation as novel Myc post-translational modifications, with different functional properties. This opens a completely unexplored field of investigation in Myc biology and suggests symmetrically dimethylated Myc species as novel diagnostic and prognostic markers and druggable therapeutic targets for GBM. It regulates cell proliferation, metabolism, stemness maintenance1 and binds to 10% of genomic loci, usually amplifying transcription from already active genes3. Myc expression and function are deregulated in a wide variety of cancers, including glioblastoma multiforme (GBM)5. Despite more than a decade of advances in surgery and chemo-/radiotherapy, GBM treatment options are disconcertingly very poor and patients life expectancy is very limited (14.6 to 18 months from diagnosis)6. GBM poor prognosis and deadly outcome seem to rely on the presence of tumour initiating cells, which share many molecular and biological features with embryonic and adult stem cells and thus are called glioblastoma stem cells (GSCs). They are difficult to eradicate by surgery and are resistant to conventional radio- and chemotherapy, allowing tumour to recur overtime7. They also present the so-called “Myc signature”8, emphasizing the essential role Myc plays in maintaining GSC properties.The Myc (c-myc) oncogene is a transcription factor required for pivotal cell functions9, acetylation10, sumoylation11, ubiquitination and consequent degradation by the proteasome machinery12. These modifications have a precise role in modulating Myc activity and have been suggested as potential therapeutic targets10.Myc is regulated by a series of post-translational modifications: phosphorylation13. Three distinct types of methylated Rs occur in mammalians. Asymmetrically dimethylated arginine (ADMA) is the most frequent, followed by symmetrically dimethylated (SDMA) and monomethylated arginine (MMA). The enzymes responsible for R methylation are the Protein Arginine Methyltransferases (PRMTs). Six proteins have been reported to undoubtedly possess the R methyltransferase catalytic domain . PRMT2, 7 and 9 have sequence similarities with the other PRMTs and are supposed to possess an equivalent enzymatic activity13. Depending on the position of the methyl groups on R terminal guanidino nitrogens, PRMTs are, basically, classified into two types: type I PRMTs  are responsible for MMA or ADMA whereas the type II PRMT5 catalyzes MMA and SDMA13. PRMT1 is the major methyltransferase in mammalian cells. It has a wide variety of substrate specificity, from histone to non-histone proteins14. From a transcriptional point of view, it is known as an activator, since the occurrence of asymmetrically (AS) dimethylated R3 on histone H4 residues (H4R3me2as) is associated with transcriptionally active chromatin15. PRMT1 is deregulated in a wide variety of cancer types, e.g. pancreatic adenocarcinoma16, gastric17 and lung cancer18. It controls the epithelial-mesenchymal transition (EMT) in cancer cells19, while playing a major role in muscle20 and B cell differentiation21. PRMT5 has received a greater attention and has been suggested as a putative druggable target in a wide variety of tumours, including GBM22. Like PRMT1, it has multiple nuclear and cytoplasmic targets and catalyzes the symmetrical (S) dimethylation of H4R3 and H3R8, usually repressing transcription23, and of H3R2, activating transcription24. PRMT5 is involved in a variety of cellular processes, such as neurogenesis25, myogenesis26, somatic cell reprogramming27 and GSCs self-renewal28. Interestingly, PRMT1 and PRMT5 have been shown to interact in GBM cells, being PRMT1 the major responsible for the activation of cancer-related genes29. They also catalyze the S- and AS-dimethylation of NMyc31 and, very recently, it has been shown that PRMT1 is required for p300 recruitment to Myc-regulated gene promoters32.Methylation of arginine (R) residues is the most common methylation event in mammalian cells, which modifies protein interacting properties, ruling a large amount of signaling pathways33. Here we show that PRMT1 associates with Myc/PRMT5 in both HEK293T cells and GSCs and that Myc is both S- and AS-dimethylated by PRMT5 and PRMT1, respectively, both in vitro and in living cells. At the functional level, S-dimethylation protects Myc from degradation, while AS-dimethylation ensure Myc proper turnover. Finally, the inhibition of either PRMT1 or PRMT5 activity affects Myc recruitment at promoters and has a profound effect on GSCs biological functions, such as neurospheres formation and differentiation capacity. These findings represent the first demonstration in GSCs of the presence of differentially dimethylated Myc species, with distinct properties, opening a completely new field of investigation in Myc-dependent GBM biology. Further, they support the hypothesis that acting on S-Myc post-translational modification may represent a possible route to control its function.We previously demonstrated that PRMT5 interacts with Myc in glioblastoma cells, including GSCs, and that PRMT5 participates, at least in part, in the activation of Myc target genes33) and associates with PRMT5 in both HEK293T and glioblastoma cells33. Since PRMT5 and PRMT1 were found associated in GBM cells29, we sought to determine whether Myc was able to promote also AS-dimethylation of R3 on histone H4 (H4R3me2as). To this aim, HEK293T cells were transfected with either a Flag-tagged Myc construct (FlagMyc/HEK293T) or an empty vector and the level of H4R3me2as was detected by western blot. Figure 34 transduced with a lentiviral, doxycycline inducible shRNA against Myc (shMyc), the level of H4R3me2s were reduced, while H4R3me2as increased ‐1,4‐pentadien‐3‐one, hereafter named inhibitor 9 (9i), which specifically inhibits PRMT1 at this concentration35. Thereafter, immunoprecipitation experiments were performed with SYM10 and ASYM24 antibodies. Figure Moreover, protein extracts from HEK293T/FlagMyc cells were reciprocally immunoprecipitated by using SYM10 or ASYM24 or anti Flag antibodies. Western blots were performed with anti-Flag or SYM10 and ASYM24 antibodies. Figure 31. We observed that upon PRMT5 interference Myc expression decreased, while in PRMT1-depleted cells, Myc levels slightly increased , bound by Myc in GSCs34 together with the Glial Fibrillary Astrocytic Protein (GFAP) promoter38, were tested. In stem conditions, either PRMT5 or PRMT1 inhibition did not affect Myc binding on CCND1 and EGFRvIII promoters. Conversely, SOX2 promoter was sensitive only to PRMT1 inhibition, whereas, either PRMT5 or PRMT1 inhibition decreased Myc binding on GFAP promoter. Interestingly, Myc detached from all these promoters in differentiating control cells. PRMT5 inhibition had no effect on Myc recruitment in these experimental conditions, whereas 9i treatment restored Myc binding above control levels only on CCND1 and EGRvIII promoters. Altogether, these data address a role to both PRMT5 and PRMT1 catalytic activity in regulating Myc loading at promoters.To evaluate the effect of PRMT1 and PRMT5 on Myc binding at its specific chromatin loci in GSCs, ChIP experiments were performed, either in stem or differentiation conditions, in the presence or absence of EPZ015666 or 9i or control vehicle ted Fig. . Therefo 39 Fig. . Also Tu 39 Fig. , whereas 39 Fig. , as alsoved Fig. . In summ41. Specifically, Myc and PRMT5 have been proved as master regulators of GSC pluripotency28, while PRMT1 loss does not affect typical pluripotency markers42. Myc interacts with PRMT533 and PRMT1 is required for p300 recruitment at Myc-regulated gene promoters32. Further, PRMT1 and PRMT5 interact in GBM29. However, no direct association between Myc, PRMT1 and PRMT5 has been reported. Here we show, for the first time, that Myc, PRMT5 and PRMT1 form a ternary complex, both in recipient HEK293T cells and GSCs , 1% Glutamine, 1% Penicillin/Streptomycin and 2% Foetal Bovine Serum .HEK293T and mesenchymal Brain Tumour 168 Neural Stem Cells2‐4‐hydroxyphenyl)‐1,4‐pentadien‐3‐one (9i) at 3 μM35. Control solvent was represented by dimethylsulfoxide . For GSCs differentiation and neurospheres formation experiments, drugs were refreshed every 48 hrs. Cyclohexymide and MG132 (Merck) were 10 μg/ml and 10 μM, respectively.EPZ015666 (Merck) was administered at a concentration of 5 μM and the PRMT1 inhibitor 1,5‐bis, anti-H4R3me2as , anti-PRMT5  and anti-PRMT1  antibodies were from Abcam . Anti-Myc  was from Santa Cruz Biotechnology . Anti-actinHRP  was from Merck. Anti-SOX2 , SYM10  and ASYM24  antibodies were from Millipore . Anti-Flag antibody  was from Merck. Secondary anti-rabbit IgG  and anti-mouse IgG  were from Merck. In most cases, filters were directly cut after the blocking procedure, according to the molecular weight of the proteins of interest. Equipment and settings: images were captured with a ChemiDoc XRS+ Gel Imaging System  and the band intensity of western blot analysis was quantified using ImageJ software. When useful, contrast/brightness of western blot images were slightly adjusted with the Power Point software.Cells were directly lysed in Laemmli buffer and western blots were performed as described33, by using 4 μg of each antibody or control IgGs . To avoid IgGs detection, either ImmunoCruz IP/WB Optima B, C, F, E  or the VeriBlot reagent  were used in western blot, after the immunoprecipitation procedure, according to the manufacturer’s instructions.For immunoprecipitation experiments, cells were lysed in 140 mM NaCl, 10 mM Tris pH 7.6, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA, protease and phosphatase inhibitors. Cells were incubated on ice for 30′, then centrifuged for 20′ at 4 °C, at 13000 rpm. Thereafter, supernatant (1–2 mg) underwent immunoprecipitation as described33, pCMVPRMT5-6myc (courtesy of dr. Levi A.) and pCMV6PRMT1-myc-Dkk , together with the corresponding empty vectors. The shMyc insert was subcloned from the KRAB lentiviral vector into the Tet-on pSLIK lentivirus33. Lentiviral production and infection were performed as described33.Plasmid transfections were performed by using Lipofectamine 2000  according to the manufacturer’s instructions. Plasmids were: pCBSFlagMycsiPRMT5, siPRMT1  and scrambled siRNAs  were transfected by using the DHARMAFect reagent (Dharmacon), according to the manufacturer’s instructions.in vitro methylation assays, HEK293T cells  were transfected with 50 nM siRNAs (or scrambled siRNA) and the day after with either PRMT5-6myc or PRMT1-myc-Dkk plasmids or control vectors (10 μg). 48 hrs later, cells were lysed in 20 mM Hepes pH 7.9, 137 mM NaCl, 20% Glycerol, 1% Nonidet P40, 2 mM EDTA, protease and phosphatase inhibitors. Cell extracts were kept in rotation for 30′, at 4 °C and then centrifuged at 12000 rpm for 20′ at 4 °C. Cell extracts were pre-cleared with 5 μg of control IgGs, for 1 hr, on ice; thereafter, 20 μl of equilibrated protein A/G Plus agarose beads  were added and the mixture was kept in rotation for 30′, at +4 °C. and then centrifuged at 10000 rpm, at 4 °C. The supernatant was incubated with either anti-Myc antibody (Santa Cruz Biotechnology), or control IgG, for an overnight, at 4 °C (to purify either overexpressed PRMT5-6myc or PRMT1-myc-Dkk). The day after, 40 μl of equilibrated protein A/G Plus agarose beads were added and the mixture was incubated in rotation for 4 hrs, at 4 °C. Beads were extensively washed with lysis buffer by centrifuging at 3000 rpm, at 4 °C. Beads and bound PRMT5-6myc or PRMT1-myc-Dkk were resuspended in assay buffer  and 500 ng of human recombinant c-Myc protein  plus 1 mM of S-adenosylmethionine (Merck) were added. The reaction mixture (100 μl) was incubated for 1 hr, at 35 °C. Thereafter, 400 μl of assay buffer were added, and dimethylated Myc was immunoprecipitated by incubating the reaction mixture with 4 μg of either SYM10 or ASYM24 antibody (Millipore), for an overnight, at 4 °C. The day after, 40 μl of equilibrated protein A/G Plus agarose beads were added and the mixture incubated for 4 hrs, in rotation, at 4 °C. After three washes in assay buffer, 20 μl of Laemmli buffer 2X were added and the immunocomplexes boiled for 5′. Dimethylated Myc was revealed by western blot.For 33. Anti-Olig2 , anti- TuJI , anti-Oct4  antibodies were from Santa Cruz Biotechnology; anti-SOX2  and anti-Nanog  were from Millipore. Anti-GFAP , anti-H4R3me2s  and anti-H4R3me2as  were from Abcam. Secondary antibodies were donkey anti-rabbit IgG Alexa Fluor 555  and goat anti-mouse Alexa Fluor 488 , both from Thermo Fisher Scientific.Immunofluorescence analyses were performed as describedvs peak to eliminate doublets and forward vs side scatter to exclude debris. The percentage of cells in the different cycle phases and in subG1 (composed of dead cells and debris) was calculated using FACS Express5 Flow Research Edition software.Cells were dissociated, counted, resuspended in PBS and then fixed in 70% ethanol for a minimum of 24 hrs. After three washes in PBS, cells were incubated with 50 μg/ml Propidium Iodide (Merck) plus 0.1 U/L RNAse (Merck) for 3 hrs at room temperature before FACS analysis by Coulter Epics XL flow cytometer . Cells were gated using both area 5 GSCs were plated in duplicate into a six-well multi-plate. Cells were allowed to grow for 10 days, in the presence or absence of EPZ015666 or 9i or control vehicle. Neurospheres formation was monitored daily by a phase contrast microscope. Images were captured with the ZOE Fluorescent Cell Imager (Bio-Rad).3 × 1033.Real time PCR were performed as described46.Chromatin immunoprecipitations were performed as describedChIPs oligonucleotides were:hEGFRv3prom_F GGCTGTTTGTGTCAAGCCTTTAhEGFRv3prom_R TTGAAGCCAATGTGTGAAGCAhSOX2prom_F AACGGACGTGCTGCCATThSOX2prom_R TCCCATTGTCCCGACGTAAAhGFAPprom_F TGGGAATGAGGCCTAGTAGGAAhGFAPprom_R GGGAATTTGGCGAAGAATGAhCCND1prom_F GCTTTCCATTCAGAGGTGTGTTThCCND1prom_R CTACCTTGACCAGTCGGTCCTTGFAP oligonucleotides used in real time PCR experiments were:GFAP_F ACCAGGACCTGCTCAATGTCGFAP_R ATCTCCACGGTCTTCACCACAll data are expressed as mean ± SEM, and significance  was calculated using two-tailed paired Student’s t test; p values < 0.05 were considered statistically significant.Supplementary info


Glucose oxidase in the presence of glucose generated H2O2, which acted as initiator of polymerization reaction. The influence of polymerization bulk composition and pH on the formation of PANI- and Ppy-based nanocomposites was investigated spectrophotometrically. The highest formation rate of PANI- and Ppy-based nanocomposites with embedded glucose oxidase and gold nanoparticles  was observed in the solution of sodium acetate buffer, pH 6.0. It was determined that the presence of AuNPs or AuCl4− ions facilitate enzymatic polymerization of aniline and pyrrole.Several types of polyaniline (PANI) and polypyrrole (Ppy) nanocomposites with embedded glucose oxidase (GOx) and gold nanoparticles (AuNPs) were formed by enzymatic polymerization of corresponding monomers (aniline and pyrrole) in the presence of 6 and 13 nm diameter colloidal gold nanoparticles (AuNPs Intensive studies and the application of conducting polymers (CPs), such as polyaniline (PANI) and polypyrrole (Ppy), have been developed extensively over the past few decades ,2,3,4,5.2O2, which is produced during glucose oxidase catalysed oxidation of glucose [Usually, CPs are synthesized by chemical ,22,23,24 glucose ,9,28,31. glucose ,20,30,31Hybrid materials based on CPs containing inorganic nanoparticles  have unique properties and, therefore, they are promising for the application in electronics, optoelectronics and in bio-devices ,35,36,37In this research enzymatic polymerization based formation of polyaniline and polypyrrole nanocomposites with embedded glucose oxidase and gold nanoparticles is described. Polyaniline and polypyrrole based composite formation rate in the presence of different AuNPs, at different pHs of solution was evaluated by UV/Vis spectroscopy. Aspergillus niger, 201 units mg−1 protein) and D-(+)-glucose were purchased from Fluka  and Carl Roth GmbH+Co.KG , respectively. Before the investigations glucose solution was allowed to stay overnight for the formation of equilibrium between α and β optical isomers. Tetrachloroauric acid (HAuCl4·3 H2O) was obtained from Alfa Aesar GmbH&Co KG , tannic acid – from Carl Roth GmbH + Co  and sodium citrate – from Penta . All solutions were prepared using deionized water purified with water purification system Millipore S.A. . The solution of sodium acetate (SA) buffer (0.05 mol L−1 CH3COONa·3H2O) with 0.1 mol L−1 KCl was prepared by mixing of sodium acetate trihydrate and potassium chloride, which were obtained from Reanal  and Lachema . Aniline and sodium hydroxide (NaOH) were purchased from Merck KGaA , pyrrole – from Acros Organics  and hydrochloric acid (HCl) – from Sigma-Aldrich . All chemicals used in experiments were either analytically pure or of highest quality. Polymers were filtered before each measurement through 5 cm column filled by Al2O3 powder to remove coloured components. All solutions were stored between measurements at +4 °C. The synthesis of AuNPs(6nm) and AuNPs(13nm) was performed according to methodology reported previously [Glucose oxidase  or AuNPs(13nm) or 0.6 mmol L−1 of HAuCl4. Formed nanocomposites were separated from the synthesis solution by a centrifugation , then they were 2 times washed with 0.05 mol L−1 sodium acetate, pH 6.0 and were collected by centrifugation. Separated and washed PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites were re-suspended in SA buffer, pH 6.0 and used for further investigations, which were performed at room temperature. PANI/AuNPs-GOx and Ppy/AuNPs-GOx composites were formed at room temperature (+20 ± 2 °C) in darkness in solution containing 0.05 mol L4−) were evaluated after enzymatic formation of nanocomposites in 0.05 mol L−1 SA buffer, pH 6.0, 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline or pyrrole, 0.75 mg mL−1 of GOx and 26.0, 3.6 nmol L−1 of AuNPs(6nm) or AuNPs(13nm) or 0.6 mmol L−1 of HAuCl4.The presence of gold in the polymerization solution, the pH of medium and the time of enzymatic polymerization have an influence on the synthesis of polymer’s nanoparticles. The investigations of the influence of AuNPs or chloroaurate ions , AuNPs(13nm) or AuCl4− was studied at different pHs ranging from 1.0 to 12 after 2 days. 0.1, 0.01 or 0.001 mol L−1 of HCl, 0.05 mol L−1 of SA buffer, pH 6.0, and 1 × 10−6, 1 × 10−4 or 0.01 mol L−1 of NaOH solutions were used for the preparation polymerization bulk solutions in pH range between 1.0 and 12.0. The duration of polymerization reaction was from 5 hours until 12 days in 0.05 mol L–1 SA buffer, pH 6.0, 0.05 mol L−1 of glucose, 0.50 mol L−1 of aniline or pyrrole, 0.75 mg mL–1 of GOx and 26.0 nmol L–1 of AuNPs(6nm) or 0.6 mmol L−1 HAuCl4. The polymerization in bulk solution consisting 0.05 mol LThe polymerization course was evaluated by UV/Vis spectroscopy. Optical absorbance of PANI/AuNPs-GOx and Ppy/AuNPs-GOx solutions was monitored by UV/Vis spectrometer Lambda 25  at the wave length range of 233–1000 nm. The investigations were performed at room temperature in plastic disposable cuvettes of 1 cm optical path length by the registration of UV/Vis spectra. After 5–10 minutes after the engagement of polymerization reaction at +20 ± 2 °C optical absorbance of PANI/AuNPs-GOx and Ppy/AuNPs-GOx solutions was registered. SigmaPlot software 12.5 was used to evaluate the data of UV/Vis measurements. The main steps of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposite formation during enzymatic polymerization are presented in CPs such as polyaniline and polypyrrole are often used in catalysis ,20 or in(6nm) and AuNPs(13nm) in SA, pH 6.0, any optical absorbance peaks were registered in vis-spectra. Although, in the presence of AuCl4− instead of AuNPs the solution of pyrrole almost instantly becomes intensively black, what is clear evidence of polymerization reaction. As it is seen from (6nm), AuNPs(13nm) or HAuCl4 become brown (in the presence of PANI) and black (in the presence of Ppy) sediments. Such colours are the most characteristic for the delocalised π-electron systems of CPs [After the mixing of all reagents used for PANI and Ppy formation in the presence of AuNPss of CPs . λ = 285 nm and λ = 570 nm if AuNPs(6nm) are added into polymerization bulk solution and only one wide peak at λ = 550 nm appears if AuNPs(13nm) are added into polymerization bulk solution. If polymerization is performed in the presence of AuCl4− the peak maximum appears at λ = 450 nm. This fact is an agreement with other researches [λ = 330 nm appears due to π-π* transition of the benzenoid ring and another peak at λ = 440 nm—due to polaron π-transition [λ = 330 nm is indication of 5,10- dihydrophenazin formation, which is cyclic aniline dimer [π-π* transition is associated with the delocalization of electrons from benzene rings on nitrogen atoms of aniline [λ = 436 nm. It is due to electron‘s excitation of benzenoid highest occupied molecular orbital to quinoid lowest unoccupied molecular orbital and indicates the formation of emeraldine forms of PANI [The intensity of colour of synthesized polymers also depends on the pH of initial polymerization bulk solution. The range of tested pHs was between 1.0 and 12. As it is seen from  if AuNPsnm are adsearches ,37. As i if AuNPsnm are adansition . Resultsne dimer . π-π* tr if AuNPsnm are ad aniline ,29. The  if AuNPsnm are ad if AuNPsnm are ad of PANI ,24,29. T of PANI .λ = 570 nm. By the increase of pH value of polymerization solution, the polaron-related peak at λ = 420 nm gradually disappears and a strong absorbance due to excision transition of the quinoid rings at λ = 560–600 nm is emerging. At the same time in strong alkaline polymerization solution optical absorbance at λ = 257 nm and λ = 320 nm, which is observed due to ð-ð* transitions of the benzenoid rings, has increased [λ = 442–570 nm and the peak at λ = 450 nm in the presence of AuNPs and AuCl4–, respectively, were evaluated to assess the formation of PANI/AuNPs-GOx nanocomposites during the polymerization. Synthesized PANI/AuNPs-GOx nanocomposites were formed in the E-PANI, which has been confirmed by the appearance of brown colour.As it is seen from spectra presented in ncreased . The abs+ ions are produced during polymerization reaction. As seen from the presented curves ((6nm) were present in the polymerization solution; and from 0.256 to 0.223 a.u. in the case where AuNPs(13nm) were present in the polymerization solution and from 0.813 to 0.283 a.u. in the case where AuCl4− was present in the polymerization solution. Synthesis of some branched polyaniline chains is expected due to polaron transitions, which are reflected in spectra [(6nm) or AuCl4−, respectively. When the pH of polymerization solution containing AuNPs(13nm) was equal to pH 10, then optical absorbance decreased until 0.217.The influence of pH value of polymerization solution on synthesized PANI/AuNPs-GOx nanocomposites is presented in d curves E, the in spectra . When th4− in pH range from 2.0 to 12. Meantime, the auto-polymerization of PANI in the presence of AuNPs(6nm) and AuNPs(13nm) was observed only in pH ranges from 1.0 until 3.0. The auto-polymerization of aniline in polymerization solution of pH 2.0 with AuNPs(6nm), AuNPs(13nm) or AuCl4− was 1.83, 1.79 and 2.78 times slower than that in polymerization solution with the same gold-based compounds and GOx. For enzymatic formation of PANI/AuNPs-GOx nanocomposites neutral polymerization solution is required due to best enzymatic activity and stability of GOx [In research performed by another authors it was predicated that aniline in polymerization bulk solution remain mobile and the synthesis of polymers could be proceeded without any enzyme . Moreove4− ions were intensively black coloured and solutions above a sediment consisting of aggregated Ppy/AuNPs-GOx become dark green. Dark colour of formed Ppy-based nanocomposites is characterized for advanced π-conjugated system [λ = 265 nm, λ = 312–361 nm, λ = 414 nm and λ = 560 nm, what is in an agreement with spectra of chemically synthesized polypyrrole [λ = 265 nm is associated with the π-conjugated structure of pyrrole rings, at λ = 312–361 nm and at λ = 414 nm—with π-π* transition of the polypyrrole‘s chain [λ = 560 nm is attributed to a transition from the valence bond to the antibonding polaron- or bipolaron-state and reveals that ultra-small clusters of AuNPs and Ppy are formed [λ = 265 nm and λ = 312–361 nm was changed just slightly but at λ = 414 nm and λ= 560 nm changes of optical absorbance were very significant. The absorbance at λ = 414 nm is characteristic for the early stage of pyrrole oxidation.The influence of pH value of polymerization solution on optical absorbance during the formation of Ppy/AuNPs-GOx nanocomposites was investigated in the next stage of our investigations. During 2-days lasting enzymatic formation of Ppy/AuNPs-GOx nanocomposites at pH values ranging from 1.0 until 12 in the presence of AuNPs or AuCld system . As it iypyrrole ,33,34. T‘s chain ,33,34. Te formed ,33,34. Bλ = 480 nm has been changed from 0.230 to 0.403 a.u. in the presence of AuNPs(6nm), from 1.33 × 10−3 to 0.141 a.u. – of AuNPs(13nm) and from 0.248 until 0.608 a.u.– of AuCl4−. In polymerization solution of pH 12, the optical absorbance at λ = 480 nm decreased down until 0.169, 3.04 × 10−3 and 0.144 a.u. in the case of AuNPs(6nm), AuNPs(13nm) and AuCl4−, respectively. Such decrease of optical absorbance by increasing pH until 12 is an agreement with investigations of Ppy chemical synthesis in polymerization bulk solution without any gold-based compounds [As it is seen from ompounds .4− into Au0 occurs very quickly and simultaneously darkening of the solution is observed [4− ions and in the absence of GOx was observed at pHs ranging from 2.0 to 3.0 during the formation of Ppy/AuNPs(6nm) and Ppy/AuNPs(13nm) nanocomposites and from 1.0 to 3.0 during the formation of Ppy/AuNPs(AuCl4−) nanocomposites. The auto-polymerization of pyrrole in the presence of AuNPs(6nm), AuNPs(13nm) and AuCl4− in polymerization bulk solution at pH 2.0 was 5.47, 1.31 and 2.89 times slower, if compared with the enzymatic formation of Ppy/AuNPs-GOx nanocomposites.When polymerization compounds are mixed, then the polymerization of pyrrole and the reduction of AuClobserved . Similarobserved . It was Significant differences of the optical absorbance were detected in the absence and in the presence of GOx, what revealed a high impact of the enzyme to the polymerization rate of monomers . Changes(6nm), AuNPs(13nm) or AuCl4− ions. AuNPs are forming seeds for polymerization and AuCl4− ions are acting as oxidators, which initiates the polymerization of CPs [0 forms AuNPs, which act as seeds for polymerization. In the presence of HAuCl4 the reduction of AuCl4− and the formation of oligoaniline is the dominant reaction in the polymerization bulk solution [4− ions and PANI is following by next steps. First of all, polyaniline coordinates Au(III) via an amine N, which is substituting Cl− in AuCl4−, because four Cl− anions are released into polymerization solution during PANI formation [2− ion is able to coordinate with an imine N of the quinoid group through electron donation from N to Au and formation of N–H----Cl− type hydrogen bonds, therefore AuCl4− ion is taking active and important role in the formation of PANI/AuNPs based nanocomposites [Enzymatic formation of PANI/AuNPs-GOx and Ppy/AuNPs-GOx depends on source of gold, which was AuNPsn of CPs  and formm), AuNPsnm or AuCm), AuNPsnm or AuCormation . Then, Amposites .−1 sodium acetate buffer, pH 6.0, which was chosen as the most optimal for mentioned gold/polymers enzymatic polymerization. Higher value of optical absorbance of polyaniline was characterized for polymerization solution with AuCl4− and achieved 0.674 a.u. ((6nm) and AuNPs(13nm) the polymerization rate of polyaniline formation was very similar and optical absorbance of 0.274 and 0.256 a.u was registered, respectively. In the presence of AuCl4− the formation of PANI/AuNPs(AuCl4−)-GOx nanocomposites was 2.46 and 2.63 times faster than that in polymerization bulk solution containing AuNPs(6nm) and AuNPs(13nm). The same influence of gold-based compounds was observed during the polymerization of pyrrole. As it is presented in (AuCl4−)-GOx nanocomposites (0.608 a.u.) was 1.51 and 4.31 times higher if compared with results obtained in polymerization bulk solution containing AuNPs(6nm) (0.403 a.u.) and AuNPs(13nm) (0.141 a.u.). It is seen that the formation rate of PANI/AuNPs(AuCl4−)-GOx and PANI/AuNPs(13nm)-GOx nanocomposites was 1.11 and 1.82 times higher if compared with of Ppy/AuNPs(AuCl4−)-GOx and Ppy/AuNPs(13nm)-GOx ((6nm)-GOx formed nanocomposites colloidal solution was 1.47 times lower than that of Ppy/AuNPs(6nm)-GOx nanocomposites colloidal solution. It could be explained by short duration of polymerization.The influence of gold compounds and polymerization rate of polyaniline and polypyrrole nanocomposites formation was investigated in 0.05 mol L674 a.u. . However3nm)-GOx . Meantim4(AuCl−)-GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites was 2.14 and 3.52 times higher if compared with that registered for PANI/GOx (0.315 a.u) [(6nm)-GOx and PANI/GOx was very similar; for Ppy/AuNPs(6nm)-GOx was 2.33 times higher than that obtained for Ppy/GOx nanocomposites. The formation rate of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites in comparison to that of PANI/GOx and Ppy/GOx is significantly higher, because AuNPs speed-up the agglomeration of formed oligomers and the most probably facilitate polymerization reaction [(6nm) and AuCl4− were chosen, which were both considered to be the most suitable for the formation of these aniline- and pyrrole-based nanocomposites [During control experiments we tested: (i) the polymerization bulk solutions of PANI and Ppy, which did not contain AuNPs and (ii) the solution, which contained glucose, GOx, and AuNPs but did not contain any monomers (aniline or pyrrole), which are required for the formation of polymers. As it is presented in 315 a.u)  and Ppy/reaction ,37. As imposites .(6nm) or 0.6 mmol L−1 AuCl4−, correspondingly. Visible aggregation of polyaniline and polypyrrole in the presence of AuNPs and AuCl4− were observed after 2 and 4.5 days of enzymatic polymerization. One of the most important parameters of polymerization process is the duration of the polymerization. Thus, the variation of duration of enzymatic polymerization enabled to achieve the optimal polymerization performance. The spectrometric investigation of enzymatic polymerization duration on formation of PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites was performed in SA buffer, pH 6.0, in the presence of aniline or pyrrole and of AuNPsλ = 450 nm was achieved after 2 and 1 days for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites formation. This optical absorbance band appears due to polaron transition from benzenoid group at highest occupied molecular orbital to quinoid group at lowest unoccupied molecular orbital, which indicates presence of emeraldine form of PANI/AuNPs-GOx nanocomposites and is in agreement with results by another authors [As it is presented in  authors ,24,27,30th day of polymerization. It is indicated, that the highest value of optical absorbance for PANI/AuNPs(6nm)-GOx (1.05 a.u.) and PANI/AuNPs4(AuCl−)-GOx (1.24 a.u.) was achieved after 10 days lasting enzymatic polymerization. The value of optical absorbance was 11.1 and 3.17 times higher than that after 5 hours lasing polymerization, respectively for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx. Moreover, long polymerization’s duration enables to synthesize larger PANI/AuNPs-GOx nanocomposites. Therefore, for the formation of smaller nanocomposites short duration of enzymatic polymerization is recommended. As it is seen from (6nm) and 0.6 mmol L−1 AuCl4− optical absorbance at λ = 450 nm increased from 0.095 to 0.517 a.u. and from 0.391 to 1.11 a.u, respectively. The increase of optical absorbance by 5.44 and 2.84 times after 5 days for PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx formed nanocomposite colloidal solutions was observed. The appearance of this peak in the presence of small nanocomposites in polymerization solution could be explained by fast movement of free electrons to the surface of AuNPs-based nanocomposites, during which electrons are scattered and quickly lose the coherence. In other words, the surface plasmon resonance bandwidth increases by the decrease of AuNPs size [As it is seen from NPs size .(6nm) or 0.6 mmol L−1 AuCl4− to the reactants UV/Vis spectra -GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites were achieved at λ = 550 nm and λ = 590 nm, respectively. After one day of enzymatic polymerization, these waves almost disappeared and new bands of π-π* transition of Ppy/AuNPs chain and a polaron transition similar to that of an ordinary Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx at λ = 448 nm and λ = 460 nm were observed more clearly [(6nm) or 0.6 mmol L−1 AuCl4− in polymerization bulk solution, a dark pyrrole was relatively rapidly synthesized and optical absorbance of formed nanocomposite colloidal solution, which was measured at two polymerization periods (after 5 h and 10 days) increased from 0.137 to 0.781 a.u. and from 0.159 to 1.05 a.u., respectively. It is seen that optical absorbance, which is characteristic for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl-)-GOx nanocomposites, after 10 days of polymerization was 5.70 and 6.60 times higher than that registered after 5 hours. This indicates the formation of polypyrrole oligomers [(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx nanocomposites has changed optical absorbance of polymerization bulk solution until 0.494 and 0.538 a.u, respectively. It was determined that after 4.5 days of enzymatic polymerization the value of optical absorbance for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl-)-GOx nanocomposites increased by 3.61 and 3.38 times, respectively. The decrease of absorbance value after 12 days of formation of nanocomposites is explained by aggregation of nanocomposites due their limited solubility.Enzymatic polymerization of pyrrole started very fast and after 5 hours of initiation of polymerization by addition of AuNPs spectra D,E showe clearly ,28,33,34 clearly . It was ligomers . From 5 (6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites was 1.05 and 2.06 times higher compared with that of Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx.It was demonstrated that synthesized PANI/AuNPs-GOx and Ppy/AuNPs-GOx nanocomposites were similar to that obtained by chemical or electrochemical methods ,33,34 anV) was evaluated using formula V = (tgα), where tgα was calculated from (6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx; 1.73 and 2.23 a.u. for Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx) formed when 100% of the monomers present in the solution are converted into polymers. Initial polymerization duration in polymerization bulk solution containing aniline with AuNPs(6nm) or AuCl4− was determined as 0.0026 and 0.0022 mol L−1 day−1, respectively; for solution containing pyrrole with AuNPs(6nm) or AuCl4− – 0.0031 and 0.0029 mol L−1 day−1, respectively. Initial formation rate of PANI/AuNPs(6nm)-GOx and PANI/AuNPs4(AuCl−)-GOx nanocomposites was 1.19 and 1.32 times slower than that of Ppy/AuNPs(6nm)-GOx and Ppy/AuNPs4(AuCl−)-GOx, respectively. However, AuNPs do not form the uniform and well interconnected structure because nanocomposites are embedded between polymer backbone [The polymerization rate  and 0.6 mmol L−1 of AuCl4− are the most suitable for the enzymatic polymerization of PANI- and Ppy-based nanocomposites. The highest rate of PANI/AuNPs-GOx and Ppy/AuNPs-GOx formation was observed in the solution of SA buffer, pH 6.0. The most optimal duration of the enzymatic synthesis of nanocomposites was within 4.5 days. In present our research enzymatically formed nanocomposites are attractive, because any other chemical compounds except glucose, GOx, AuNPs or AuCl4− and corresponding monomer (aniline or pyrrole), which forms conducting polymer matrix, are used in the polymerization course.Enzymatic polymerization of aniline and pyrrole monomers in the presence of AuNPs and AuCl


N-[folate(polyethylene glycol)-2000 prepared by thin-film hydration. Small unilamellar vesicles were achieved by sonication and extrusion. Scanning electron microscopy evidenced round-shaped nanocapsules presenting a smooth surface, 150 nm diameter and polydispersity index <0.2, estimated by dynamic light scattering. Tarin entrapment rates were over 80% and leakage of ~3% under 40 days of storage at 4 °C. Entrapped tarin exhibited an 83% release after 6 h at pH 4.6–7.4 and 36 °C. Both free and encapsulated tarin exhibited no in vitro toxicity against healthy mice bone marrow and L929 cells but stimulated the production of fibroblast-like and large round-shaped cells. Encapsulated tarin resulted in inhibition of human glioblastoma (U-87 MG) and breast adenocarcinoma (MDA-MB-231) proliferation, with an IC50 of 39.36 and 71.38 µg/mL, respectively. The effectiveness of encapsulated tarin was similar to conventional chemotherapy drugs, such as cisplatin and temozolide. Tarin liposomal nanocapsules exhibited superior pharmacological activity compared to free tarin as a potential chemotherapy adjuvant.The search for natural anticancer agents and nanocarrier uses are a part of the current strategies to overcome the side effects caused by chemotherapeutics. Liposomal nanocapsules loaded with purified tarin, a potential immunomodulatory and antitumoral lectin found in taro corms, were produced. Liposomes were composed by 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine, cholesterylhemisuccinate, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine- Cancer is among the leading causes of death worldwide, defined by the exacerbated proliferation of normal cells into tumor cells due to a multistage process that usually involves mutation . Due to Breast cancer is the second main cause of death by cancer in women, second only to lung cancer. The American Cancer Society estimated that about 266,120 new cases of invasive breast cancer in women would be diagnosed in 2018, and that about 40,920 women would die from this disease . Most woThroughout the years, several strategies have been used to overcome or minimize side effects, including the search for natural anticancer agents, which are extensively associated to lower toxicity, due to their favorable body absorption and metabolism compared to conventional synthetic drugs . AdditioColocasia esculenta) .,38.50 foed tarin .36 μg/mLolide 33. μg/mL inN-glycans that are a part of the Lewis Y/CD174, H2/CD173, and CA125 antigens, commonly found in human cancer cells   (Lipoid GMBH) and CHEMS (cholesterylhemisuccinate) (Sigma-Aldrich Co) (5.7:3.8:0.5 μmol of lipids) in a chloroform solution under constant stirring at 150 rpm for 15 min. The organic solution was then removed by evaporation under reduced pressure for 25 min at 40 ± 1 °C and constant stirring at 120 rpm. The dry lipid film was rehydrated to reach a 0.01 M concentration in an aqueous phase consisting of 0.3 M ammonium sulfate (pH 7.4) containing tarin at 1 mg/mL, followed by incubation for 12 h at 4 °C. Then, the suspension was maintained under magnetic stirring for 40 min and the large unilamellar vesicles formed were sonicated for 1 min, followed by a 12-cycle extrusion through a polycarbonate 0.2 μm pore membrane. Unencapsulated and encapsulated tarin were recovered by ultracentrifugation carried out at 150,000× g at 4 °C for 90 min using an Optima L-90k ultracentrifuge . The pellet containing encapsulated tarin was suspended in a HEPES buffered saline solution (HBS) at pH 7.4 and the supernatant containing unencapsulated tarin was quantified by Peterson’s method [Liposomes were obtained by modifying the protocols described by dos Santos Ferreira et al.  and Andrs method  to avoids method .The liposome preparation protocol was subjected to some variations in order to optimize stability and entrapment efficiency conditions. Different organic solvents, sonication times, initial tarin loads, and times for tarin entrapment were tested .l-lysine, and fixed in 4% glutaraldehyde prepared in 0.1 M phosphate buffer pH 7.2, at 4 °C for 48 h. The coverslips were then washed three times for 5 min with the same phosphate buffer. Fixed samples were then dehydrated as follows: 35% ethanol 1× for 15 min, 50% ethanol 1× for 15min, 75% ethanol 1× for 15 min, 95% ethanol 2× for 15 min and absolute ethanol 3× for 20 min. Subsequently, samples were chemically dried by immersion in 1–2 mL of hexamethyldisilazane (HMDS) for 10 min, twice, and left in a desiccator at room temperature overnight. Dried samples were then mounted on a sample stub with a double-sided sticky tape, sputtered with gold and visualized using a JEOL JSM-6460LV scanning electron microscope (SEM)  at 20kV.Liposome characterization was performed according to Murtey and Ramasamy . LiposomSize distribution and the polidispersity index (PdI) of liposomal tarin nanocapsules stored at 4 °C for 180 days were assessed by dynamic light scattering (DLS) .Nanocapsule tarin leakage was quantified by Peterson’s method  during 4The influence of time, temperature and pH on tarin release was determined according to the protocol described by Deniz et al. . A liposAdult isogenic male C57Bl/6 mice (aged 10–16 weeks) were provided and maintained at the Animal Center Laboratory (NAL), belonging to the Universidade Federal Fluminense (UFF), Rio de Janeiro, Brazil.The research protocol was approved by the Universidade Federal Fluminense Ethics Committee in Animal Use (CEUA), under No. 821/2016.5 cells/well and 1.5 × 105 cells/well, respectively, in 100 μL of RPMI-1640 media (Sigma-Aldrich Co), supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamin, 5 × 105 M2-mercaptoethanol, and incubated at 37 °C in a humidified atmosphere containing 5% CO2. After 24 h incubation, the time required for cell adherence to the plate, different free or encapsulated tarin concentrations (from 0.78125 to 100 μg/mL) and empty liposomes were added to a final volume of 100 μL. The positive controls comprised cells cultivated in culture media, while 12.5% sodium azide (25 μL) was added to the negative control wells. Cells were incubated for an additional 24 h and cell viability was assessed by the colorimetric assay using resazurin as the indicator. A 20 μL aliquot of resarzurin 125 μg/mL was added to each well, followed by 4 h of incubation. Fluorescence intensity was determined using a SpectraMax Microplate Reader M4  at 530 nm (excitation filter), 570 nm (cutoff), and 586 nm (emission filter) wavelengths.Tarin cytotoxicity was assessed by the median inhibitory dose, capable of killing 50% of cultured cells. Healthy mouse bone marrow and L929 cell lines (Sigma-Aldrich Co) were seeded in 96-well polystyrene microplates at a concentration of 5 × 106 cells/mL) in RPMI-1640 media (Sigma-Aldrich Co), supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamin, 5 × 10−5 M 2-mercaptoethanol and 20 µg/mL gentamicin, in the presence or absence of 20 µg/mL tarin, at 37 °C in a humidified atmosphere containing 5% CO2, for 14 days. Media were replaced every five days. Cell samples were collected on days 0, 5, and 14, and transferred to glass slides by centrifugation (284× g for 10 min at room temperature) using a Cytopro 7620 centrifuge . Cells were analyzed after staining by the May–Grunwald–Giemsa method under an optical microscope  [Cells were cultured  . Photomi, Japan) .® HTB-26™) (breast adenocarcinoma) and U-87 MG (ATCC ® HTB-14™) (glioblastoma) were determined by the redox resazurin method described in 5 cells/well) were seeded in 25 cm2 culture bottles, containing Dulbecco’s modified Eagle medium (Nutrient Mixture F-12 - DMEM/F-12) (Sigma-Aldrich Co), supplemented with 10% fetal calf serum (FCS), 2 mM l-glutamin, 5 × 105 M2-mercaptoethanol. Culture bottles were incubated at 37 °C in a 95% humidified atmosphere containing 5% CO2 until a semiconfluent monolayer was achieved.The viabilities of the human cancer cell lines MDA-MB-231 . Cancer cells at 5 × 105 cells per well, at a final volume of 100 μL, were cultivated in 96-well culture microplates containing DMEM medium and the cytotoxicity evaluation procedure was performed as described previously.Cells from the semiconfluent monolayer were detached from the culture bottles with soft movements after the addition of 2 mL of a solution containing 1 mL 20% trypsin, 0.5 mL 10% ethylenediaminetetraacetic acid (EDTA), and 3.5 mL PBS. Cell suspensions were then centrifuged at 200× x-axis were standardized per tarin concentration  followed by multiple comparisons by Tukey’s method. The GraphPad Prism software  was used.The methodology described herein allowed for the reproducible production of spherical, smooth-surfaced, and nanomeric (<0.2 nm) liposomal capsules that exhibit high tarin entrapment efficiencies and low leakage rates under storage conditions. Liposomal tarin nanocapsules exhibited no toxicity to healthy mice bone marrow and L929 cells and inhibited the proliferation of MDA-MB-231 and, to a higher extent, U-87 MG cell lines, indicating an improvement of the pharmacological ability of encapsulated tarin compared to its free form. Healthy mice bone marrow cells treated with free and encapsulated tarin also indicates a possible immunomodulatory effect, which could be useful to boost the host immune system. Liposomal tarin nanocapsules seem to be a promising anti-cancer candidate, obtained from a food matrix and exhibiting two of the most valuable characteristics for any anticancer drug, namely anti-tumor efficacy and low toxicity to healthy cells, enabling it as a putative composite to be associated as an adjuvant to traditional chemotherapy.


The transcription of heat shock protein (HSP) 27, HSP40, HSP60, HSP70 and HSP90 mRNA was tested by fluorescence quantitative PCR. The results showed that the mRNA expression of HSPs exposed to 10−6 mol/L Cd showed a sustained decrease at 12–48 h exposure. A statistically significant increase in the mRNA expression of HSPs in the case of Se group was observed, as compared to the control group of chicken splenic lymphocytes. Concomitantly, treatment of chicken splenic lymphocytes with Se in combination with Cd enhanced the mRNA expression of HSPs which were reduced by Cd treatment. This indicated that the protective effect of Se against the toxicity of Cd might, at least partially, be attributed to stimulation of the level of HSPs.Cadmium (Cd) is a heavy metal that poses a hazard to animal health due to its toxicity. Selenium (Se) is an important nutritional trace element. However, the potential protective effects of Se against Cd-induced toxicity remain to be elucidated. To investigate the cytotoxicity of Cd on bird immunocytes  Splenic lymphocytes density was adjusted to 1.5 × 106 cells/mL and the viability of the freshly isolated cells was always above 95% (trypan blue exclusion test). For the monitoring of various parameters in the present investigation, the control group was incubated for 12 h, 24 h, 36 h, 48 h without reagents. 10−6 mol/L Cd (Cd group), 10−7 mol/L Se (Se group), and the mixture of 10−7 mol/L Se and 10−6 mol/L Cd (Se+Cd group) were added while cells at logarithmic growth phase and incubated for 12 h, 24 h, 36 h, 48 h, respectively. The concentrations of Cd and Se used in this study were according to our previous studies [All procedures used in this experiment were approved by the Institutional Animal Care and Use Committee of Northeast Agricultural University. Spleens were dissected from Isa brown cocks (60 days old) and were collected aseptically and placed in sterile phosphate-buffered saline . Single cell suspension was prepared by gently pushing the splenic pulp through a sterile stainless steel mesh with a pore size of 100 micron. Cells were washed and resuspended in 5 mL sterile PBS and then layered over 5 mL lymphocytes separation medium . The splenocyte preparations were enriched by centrifugation . The RNA concentrations were determined using the GeneQuant 1300. Reverse transcription reaction (40 µL) consisted of the following: 10 µg of total RNA, 1 µL of M-MLV reverse transcription, 1 µL of RNase inhibitor, 4 µL of dNTP, 2 µL of Oligo dT, 4 µL of dithiothreitol, and 8 µL of 5×buffer. The procedure of the reverse transcription was according to the manufacturer’s instructions (Invitrogen). The reverse transcription products (cDNA) were then stored at −20 °C for PCR.To design primers, we used the chicken HSP27, HSP40, HSP60, HSP70 and HSP90 mRNA GenBank sequence with accession number of NM_205290, NM_001199325, NM_001012916, NM_001006685, NM_001109785, respectively. Chicken β-actin (GenBank accession number L08165) as a housekeeping gene was used as an internal reference. Primers  were desΔCT,T/AT-ΔCT,R/AR, where ΔCT,T = Ct (HSP sample), ΔCT,R = CT (β-actin sample), AT = slope of standard curve (HSPs), AR = slope of standard curve (β-actin).Real-time quantitative reverse transcription PCR was used to detect the expression of HSP27, HSP40, HSP60, HSP70 and HSP90 gene in cells by using SYBR Premix Ex TaqTM , and real time PCR work was done in the ABI PRISM 7500 real-time PCR system . The program was 1 cycle at 95 °C for 30 s and 40 cycles at 95 °C for 5 s and at 60 °C for 34 s. Dissociation curves were analyzed by Dissociation Curve 1.0 Software (Applied Biosystems) for each PCR reaction to detect and eliminate the possible primer-dimer and non-specific amplification. The mRNA relative abundance was calculated according to the method of Pfaffl . Resultsp < 0.05) was obtained by one-way analysis of variance, further analysis was carried out. All data showed a normal distribution and passed equal variance testing. Differences between means were assessed using Tukey’s honestly significant difference test for post hoc multiple comparisons. Data are expressed as the mean ± standard deviation.Statistical analysis of all data was performed using SPSS for Windows . When a significant value (The present results indicated that Cd exposure impaired the mRNA expression of HSPs in chicken splenic lymphocytes. Moreover, the protective effect of Se against the toxicity of Cd might, at least partially, be attributed to stimulate the level of HSPs. Therefore, Se can be considered a potential therapeutic nutrient to protect against toxicity induced by Cd.