Addition of these guests result in the formation of various supra-amphiphiles with well-defined structures, thus induce the generation of different aggregates. A diverse of aggregation morphologies including nanospheres, nanorods, films, spheric micelles, vesicles and macrowires are constructed upon the influence of specific complexation, which highlights the present work with abundant control on the shapes and dimensions of self-assemblies.Control of self-assembly is significant to the preparation of supramolecular materials and illustration of diversities in either natural or artificial systems. Supra-amphiphiles have remarkable advantages in the construction of nanostructures but control of shape and size of supramolecular nanostructures is still a great challenge. Here, we fabricate a series of supra-amphiphiles by utilizing the recognition motifs based on a heteroditopic porphyrin amphiphile and its zinc complex. These porphyrin amphiphiles can bind with a few guests including Cl The supra-amphiphiles spontaneously assemble to well-defined nanostructures but control of shape and size of supramolecular nanostructures is still a great challenge. Here the authors demonstrate control over shape and size of self-assemblies by using the recognition motifs of an amphiphilic porphyrin The supra-amphiphiles, a novel research field that combining supramolecular chemistry and traditional amphiphiles, have attracted increasing attention as they can spontaneously assemble to construct a variety of well-defined nanostructures such as micelles, vesicles, tubes, nanorods and nanosheets, which show great application potential in nanodevice, memory storage, drug or gene delivery, protein probe, tissue regeneration, and ion channel regulation12. The supra-amphiphiles are a class of molecules bearing both hydrophilic and hydrophobic segments that are conjoined by noncovalent bonds or dynamic covalent bonds20 including π−π stacking24, electrostatic interaction26, hydrogen bonding28, coordination29, charge-transfer interaction30, and imine formation31. Owing to the dynamic and reversible nature of these covalent or noncovalent interactions, the self-assemblies derived from supra-amphiphiles can be readily controlled by external stimuli including temperature, concentration, solvent, pH, enzyme, redox, ionic strength, and light32. Although the previous studies exhibited the advantages of using dynamic covalent bonds or supramolecular interactions to construct amphiphiles, fine control in the shape, dimension, and size of supramolecular nanostructures is still a great challenge.Controlling formation of the self-assemblies with ordered and diversified morphologies are important for imitation of natural systems and application of artificial self-assembliesπ-conjugated architectures, excellent photosensitivity and biochemical functionality, porphyrins have been widely used as building blocks in supramolecular self-assemblies41. Functional groups can be easily introduced into the porphyrin moieties, endowing the obtained supramolecular systems with interesting properties. α,α,α,α-meso-Tetrakis(2-aminophenyl)porphyrin 43 and its derivatives possess unique “picket fence” topological structures and attractive features. They have been previously used as binucleating ligands47, building blocks for cages48, anion receptors51, synthetic models for oxygen binding hemoproteins52 and cytochrome oxidase54, as well as chloroperoxidase analogs55. The unique structure of α,α,α,α-H2TamPP makes it very suitable as a building block to prepare supra-amphiphiles for the controllable self-assembly. Herein, we design and synthesize a porphyrin-based heteroditopic amphiphile by utilizing α,α,α,α-H2TamPP. This unilateral porphyrin amphiphile and its zinc complex consist of a hydrophobic urea-derived porphyrin and four hydrophilic oligoether chains that point to the same side of the porphyrin plane, which can not only be served as receptors to bind with inorganic or organic guests including Cl–, coronene, C60, and pyridyl ligands, but also be applied in the construction of supra-amphiphiles to realize the fine control of amphiphilic self-assembly upon the addition of different guests. Addition of these specific guests result in the fabrication of supra-amphiphiles with different well-defined topological structures, thus induce the change of self-assembly morphologies.Due to their distinctive 2TamPP 1 could be easily prepared by cyclization from 2-nitrobenzaldehyde and pyrrole, reduction, and then atropisomerization56, according to the procedures described in the Methods section. The four amino groups of 1 were transformed to isocyanates with triphosgene to generate the compound 2, while reaction of p-acetaminophenol with tetraethylene glycol monotosylate 3 produced tetraethylene glycol-substituted aniline 4. The isocyanate 2 was then reacted with excess 4 in dichloromethane to furnish the amphiphilic porphyrin 5 in 68% yield. Complexation of 5 with Zn(OAc)2•2H2O in chloroform afforded the Zn-porphyrin derivative 6 in 73% yield. Both 5 and 6 were fully characterized by 1H NMR, 13C NMR, high-resolution mass spectroscopy (HRMS), infrared spectroscopy (IR), UV-vis spectroscopy, and fluorescence spectroscopy  was firstly investigated by TEM observations. As self-assemblies of most organic molecules usually behave in homogeneous solution, the heteroditopic amphiphile 5 aggregated into small spheres of several nanometers in diameter in THF /2), and the intercept is Kav. Therefore, Kav was determined to be 5.3 (±0.1) × 104 M‒1 for 52•7. Since K1/K2 = 4:1 for statistical systems (K1 = [5•7]/{[5][7]} and K2 = [52•7]/{[5•7][5]}), K1 and K2 were calculated to be 8.5 (±0.2) × 104 M‒1 and 2.1 (±0.1) × 104 M‒1, respectively. The high binding ability ensures the supra-amphiphile stable during the self-assembly. In spite of the fact that uncomplexed species usually increase at low concentrations, hydrophobic interactions existed during self-assembly in aqueous solutions would greatly enhance the stability of the supra-amphiphiles.Furthermore, it was found that the size and shape of aggregation morphology of the heteroditopic amphiphile tra Fig.  and exis5 and 7 are relatively disorganized with the existence of a few nanorods  × 103 M‒1 and 8.0 (±0.4) × 102 M‒1, respectively -1,3,5-triazine (TPT) 10 was added to a 6 solution, the central zinc ion coordinated with the N atoms on pyridyl guests to generate the supra-amphiphilic complexes 62•9 and 63•10. The coordination between 6 and the two pyridyl guests was confirmed by 1H NMR, 2D NOESY NMR, UV-vis, and fluorescence spectroscopy. Change of the chemical shifts of protons on both 6 and the pyridyl compounds  × 103 M‒1 and 4.0 (±0.2) × 102 M‒1, respectively, and K1, K2, and K3, of 63•10 in THF were 2.4 (±0.1) × 103 M‒1, 8.1 (±0.1) × 102 M‒1 and 2.7 (±0.1) × 102 M‒1, respectively. These considerable binding affinities are conducive to the formation of stable supra-amphiphiles.Pyridyl compounds are good alternatives to be used as the guests that can easily coordinate with metalloporphyrins9 was used as the guest, a dumbbell-shaped supra-amphiphile 62•9 was formed, which is composed of a stiff hydrophobic segment including two coordinated zinc-porphyrins in the core and two parts of flexible hydrophilic oligoether segments at the two sides that are exposed to the aqueous environment. TEM images showed that the supramolecular amphiphile 62•9 self-assembled into stable micelles with different sizes. As shown in Fig. 62•9 cast onto micrograph TEM grids. The observed preservation of immense spheric micelles suggests that the amphiphilic complexes stacked together with the aid of hydrogen bonding, π−π stacking, and hydrophobic interactions to form spheres  as a toolrns Fig. , the nanity Fig.  while thine Fig. . The conine Fig.  from theure Fig. , the res60, and/or Cl–. As the distance between two hydrophobic porphyrin planes enlarged by insertion of the aromatic guests with different sizes and shapes, various aggregation morphologies such as nanospheres, nanorods, and films were formed as a result of the influence of specific complexation interactions. Self-assembly of the amphiphilic Zn-porphyrin derivative could also be controlled to form spheric micelles, vesicles and macrowires after coordination with 4,4′-bipyridine or 2,4,6-tri(pyridin-4-yl)-1,3,5-triazine. The presented studies provide a new strategy to construct supra-amphiphiles by using porphyrin-based recognition motifs, and intensively exhibit the advances of supra-amphiphiles for morphological control with the convenient addition of different guests, which will have great potential applications in smart materials and biological systems.A amphiphilic porphyrin with four oligoether chains and its corresponding Zn-coordinated derivative were synthesized. The amphiphilic porphyrin can self-assemble into nanostructures, while the self-assembly morphologies changed dramatically upon the addition of coronene, C2. Other solvents were employed as purchased. NMR spectra were collected on a Bruker AVANCE DMX-500 spectrometer at room temperature. Chemical shifts (δ) are given in ppm and referenced to the standard TMS or residual solvent peaks. Mass spectra were recorded on an Agilent Technologies 6530 Q-TOF mass spectrometer with ESI resource. Dynamic light scattering (DLS) were recorded on a Malvern Nanosizer S instrument at room temperature. Transmission electron microscope (TEM) was performed at 120 kV using JEM-2010 or JEM-1010. Scanning electron microscope (SEM) was done with a JSM-6390LV or TM-1000 using second electron images. High-resolution transmission electron microscope (HRTEM) was carried out on a JEM-2100F at room temperature. Atomic force microscope (AFM) was carried out on a Bruker ICON3-sys. UV-vis spectra were measured with a Hitachi UH-5300 at room temperature. Fluorescence spectra were recorded on a Perkin Elmer LS55 at room temperature. Fourier transform infrared (FTIR) spectroscopy was carried out on a Thermo Scientific Nicolet iS50 at room temperature and only noteworthy absorptions (in cm–1) are listed.All chemical reagents were commercially available and used as received. Dichloromethane was dried and redistilled with CaH56. After the mixture was stirred for 0.5 h, 75 mL of chloroform was added. The mixture was cooled to 0 °C to precipitate the product. The solid was filtered and washed with chloroform, and then dried at 100 °C to furnish meso-tetrakis(2-nitrophenyl)porphyrin . A solution of SnCl2•2H2O  in conc. hydrochloric acid (40 mL) was added dropwise to a solution of meso-tetrakis(2-nitrophenyl)porphyrin  in conc. hydrochloric acid (100 mL) at room temperature under N2 atmosphere. The reaction was carried out for 3 h at room temperature, and was then continued at 65 °C for 0.5 h. After cooling to room temperature, NH4OH (160 mL) was added dropwise in an ice water bath, chloroform (285 mL) was then added and the mixture was stirred overnight. The organic phase was separated and the aqueous phase was extracted with chloroform (3 × 100 mL). The organic phase was combined, filtered over Celite, and then concentrated to a volume of 50 mL. After ethanol (50 mL) and NH4OH (10 mL) were added, the mixture was concentrated to a volume of 25 mL. The precipitated purple solid was filtered, washed with methanol (3 × 10 mL), and dried to give a solid containing 1 and its other three isomers. In a 250 mL three-necked flask, 36 g of silica gel (300‒400 mesh) and benzene (85 mL) were added under N2 atmosphere. After the mixture was stirred at 78 °C for 2 h, the solid containing 1 and its isomers was added and the mixture was further refluxed for 20 h. After cooling to room temperature, the dark slurry was then poured into a silica gel column. Benzene/diethyl ether = 1:1 was used to elute the three isomers of 1 and acetone/diethyl ether = 1: 1 was then used to elute 1 . 1H NMR : δ 8.80 , 7.68 , 7.52 , 7.15 , 7.00 , 4.61 , and ‒2.72 . HRESI-MS: m/z calcd for [M]+ C44H34N8, 674.2906, found 674.2902, error 0.6 ppm.Pyrrole  was added dropwise to a solution of 2-nitrobenzaldehyde  in acetic acid (500 mL) under refluxp-toluenesulfonyl chloride  in dichloromethane (50.0 mL) was added dropwise and then reacted for further 12 h at room temperature. After dichloromethane was removed under vacuum, the residue was purified by flash column chromatography with a mixture of petroleum ether and ethyl acetate as eluent  to afford the compound 3  as a colorless liquid. 1H NMR : δ 7.77 , 7.32 , 4.14 , 3.66 , 3.63–3.58 , 3.56 , 3.53–3.50 , 3.35 , and 2.42 . 13C NMR : δ 144.89, 133.15, 129.92, 128.06, 72.02, 70.67, 69.36, 68.76, 59.09, and 21.71.Triethylamine  was added to a solution of tetraethyleneglycol monomethyl ether  in dichloromethane (80 mL) and the mixture was cooled to 0 °C. A solution of 3  was added to a mixture of 4-hydroxyacetanilide , anhydrous potassium carbonate , sodium iodide (50 mg), and acetonitrile (80 mL). The mixture was stirred at reflux for 12 h. After the reaction mixture was cooled to room temperature, it was filtrated and the solid was washed with dichloromethane (50 mL). The filtrate was concentrated under vacuum and the residue was dissolved in a KOH  solution in ethanol (40 mL) and water (10 mL). After the mixture was stirred for 6 h at reflux, the solvents were removed under vacuum and the residue was purified by flash column chromatography (eluent: petroleum ether/EtOAc = 1: 3) to afford the compound 4  as a colorless oil. 1H NMR : δ 6.75 , 6.62 , 4.04 , 3.80 , 3.74–3.68 , 3.68–3.60 , 3.54–3.52 , and 3.36 . 13C NMR : δ 151.75, 140.21, 116.15, 115.77, 71.82, 70.50, 69.78, 68.05, and 58.87.The tosylate 1  and Et3N (0.6 mL) in dichloromethane (30 mL), a solution of triphosgene  in dichloromethane (30 mL) was added dropwise at 0 °C under nitrogen atmosphere. After the reaction solution was stirred for 3 h at room temperature, the intermediate 4  was added at 0 °C and the mixture was then stirred for further 2 h at room temperature. After water (30 mL) was added, the organic layer was separated, and the aqueous layer was further extracted with dichloromethane (20 mL). The combined organic solution was washed with brine (3 × 30 mL), and dried over anhydrous Na2SO4. The filtrate was concentrated under vacuum and the residue was purified by flash column chromatography (eluent:dichloromethane/methanol = 100:1) to afford the amphiphilic porphyrin 5  as a purple solid. M.p. 108‒110 °C. 1H NMR  δ 8.78 , 8.41 , 8.32 , 7.77 , 7.68 , 7.59 , 7.37 , 6.84 , 6.51 , 3.80 , 3.57 , 3.50–3.34 , 3.16 , and –2.64 . 13C NMR  δ 153.81, 153.14, 150.58, 139.60, 135.89, 132.77, 131.84, 129.57, 122.19, 121.88, 119.85, 116.23, 114.80, 99.99, 71.68, 70.30, 70.21, 70.18, 69.99, 69.34, 67.53, 58.44, and 46.24. HRESI-MS: m/z Calcd for C108H126N12O12 [M + H]+: 1975.9008, Found:1975.9080, error 3.6 ppm. IR (thin film): νmax (cm–1) = 3550, 3474, 3414, 3126, 1635, 1616, and 1400.To a solution of 5  in CHCl3 (30 mL) was added Zn(OAc)2 , and the mixture was stirred for 3 h at 60 °C. After the mixture was evaporated to remove solvents, the residue was purified by flash column chromatography (eluent:dichloromethane/methanol=100:1) to afford the amphiphilic Zn-porphyrin 6  as a purple solid. M.p. 112‒115 °C. 1H NMR  δ 8.70 , 8.42 , 7.83–7.72 , 7.38 , 6.67 , 6.32 , 3.72 , 3.55 , 3.44–3.32 , and 3.16 . 13C NMR  δ 153.12, 152.47, 149.63, 139.20, 134.81, 132.29, 132.19, 131.52, 128.50, 120.83, 119.45, 119.35, 115.36, 114.12, 71.17, 69.78, 69.70, 69.66, 69.47, 68.80, 67.00, and 57.92. HRESI-MS: m/z Calcd for C108H124N12O12Zn [M + H]+: 2037.8143, found: 2037.8174, error 1.5 ppm. IR (thin film): νmax (cm–1) = 3565, 3550, 3473, 3416, 3126, 1652, 1616, and 1400.To a solution of 5 or its Zn-porphyrin derivative 6 and the corresponding aromatic compounds (7 or 8) or pyridine templates (9 or 10) were dissolved in THF or in a mixed organic solvent and the solutions were mixed in a specific mole ratio. The mixtures were then added into the deionized water and stirred overnight until THF or other organic solvents were volatilized completely. Drops of each solution were cast on carbon-coated grids  and dried naturally for two days before TEM photography. Drops of these mixed solutions were cast on monocrystalline silicon, dried naturally for 2 days and sprayed gold before SEM photography, or cast on monocrystalline silicon and dried naturally for 2 days before AFM photography. More detailed procedures of sample preparation for electron microscope are described in Supplementary Methods.Amphiphilic porphyrin Supplementary InformationPeer Review File


Max), relative contribution of the mitochondrial respiratory chain complexes, calcium retention capacity (CRC)—a marker of apoptosis—and reactive oxygen species (ROS) production were determined using high-resolution respirometry, spectrofluorometry, and electron paramagnetic resonance in gastrocnemius muscles from both hindlimbs. IR significantly reduced mitochondrial VMax (from 11.79 ± 1.74 to 4.65 ± 1.11 pmol/s*mg wet weight (ww), p < 0.05, −50.2 ± 16.3%) and CRC (from 2.33 ± 0.41 to 0.84 ± 0.18 µmol/mg dry weight (dw), p < 0.05; −61.1 ± 6.8%). ROS tended to increase in the ischemic limb . Although tending to reduce IR-related ROS production (−42.4%), sildenafil failed to reduce muscle mitochondrial dysfunctions . In conclusion, lower limb IR impaired skeletal muscle mitochondrial function, but, despite tending to reduce ROS production, pharmacological preconditioning with sildenafil did not show protective effects.Lower-limb ischemia-reperfusion (IR) is frequent and associated with significant morbidity and mortality. Phosphodiesterase 5 inhibitors demonstrated antioxidant and beneficial effects in several organs submitted to IR, but their effects on muscle mitochondrial functions after lower-limb IR are unknown. Unilateral hindlimb IR (2 h tourniquet followed by 2 h reperfusion) without or with sildenafil (1mg/kg ip 30 minutes before ischemia) was performed in 18 mice. Maximal oxidative capacity (V Peripheral arterial disease (PAD) is frequent and associated with significant morbidity and mortality ,2. PAD oRecent improvement in the knowledge of PAD pathophysiology might be useful to open new therapeutic approaches. Thus, skeletal muscle mitochondrial dysfunctions, together with increased reactive oxygen species (ROS) production are key factors. Mitochondria play a central role in cell homeostasis, since they are the primary sites of energy production through ATP synthesis. Besides cellular energy metabolism adaptation, such organelles also modulate the production of ROS and consequently cell apoptosis, which is increased when mitochondrial calcic retention capacity (CRC) is impaired ,8,9,10. Ischemic preconditioning generally demonstrated to be protective in lower limb ischemia-reperfusion ,13. IschIn this view, phosphodiesterase inhibition deserves to be investigated. Cyclic nucleotide phosphodiesterase families (PDEs) control cyclic nucleotide levels and play a major role in the control of normal and pathological cellular signaling ,21,22. PThe aim of this study was, therefore, to investigate whether pharmacological pre-conditioning with the PDE5 inhibitor might, through its anti-oxidant effect, protect skeletal muscle mitochondrial oxidative capacity and calcium retention capacity, using the model of tourniquet-induced lower limb IR.Eighteen male Swiss mice (12–16 week old), provided by JANVIER Labs , were housed in a thermo-neutral environment at 22 ± 2 °C on a 12 h day/night cycle and were provided food and water ad libitum. The protocol was approved by the Regional Committee of Ethics in Animal Experimentation of Strasbourg (C.R.E.M.E.A.S) and the Ministry of Higher Education and Research (CREMEAS n°2018022716192465v3), and the animal care complied with the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. Washington: National Academy Press, 1996.®, BAXTER S.A.S.) and oxygen and placed on heating blankets  at 37 °C. Spontaneous ventilation was allowed through an oxygen-delivering mask, with different concentrations of isoflurane depending on the surgical stage (2% during painful stimuli and 1% during latent periods). At the end of the experiment, left and right gastrocnemius muscles were dissected and immediately immersed in Krebs solution at 4 °C for the extemporaneous analyses of mitochondrial functions.All mice were subjected to 2 h ischemia through a tourniquet placed on the right hindlimb, followed by 2 h reperfusion . The lef2 (3 mM), K lactobionate (60 mM), taurine (20 mM), KH2PO4 (10 mM), HEPES (20 mM), sucrose (110 mM), creatine (20 mM), BSA (1 g/L).Muscle oxygen consumption was determined using a high-resolution oxygraph , in 2 ml of Miro5 + Cr thermostated at 37 °C, containing EGTA (0.5 mM), MgCl0) with glutamate (10 mM) and malate (2.5 mM). Then, maximal respiration rate (VMax) was measured under continuous stirring in the presence of saturating amount of ADP (2 mM) as a phosphate acceptor. Succinate injection  allowed the activation of all complexes . Finally, ascorbate (0.5 mM) and N, N, N ‘, N’-tetramethyl-p-phenylenediamine dihydrochloride  were injected, Vasc/TMPD representing the complex IV contribution. Results were expressed as pmol/sec/mg wet weight.Permeabilized fibers were placed in chambers to record the basal oxygen consumption  was determined in permeabilized “ghost” muscle fibers [2+ needed to trigger a massive Ca2+ release by the mitochondria due to mPTP opening was calculated from a standard curve relating [Ca2+] to the fluorescence of calcium green and expressed as μmol/mg dry weight.The time to the opening of the mitochondrial permeability transition pore (mPTP) following Cae fibers . The amo2 2.5 mM, MgSO4 1.2 mM, NaHCO3 25 mM, KH2PO4 1.03 mM, D(+) glucose 5.6 mM, Na-Hepes 20 mM, deferoxamine 25 µM, and DETC 5 µM. Tissues were cut into 1 to 2 mm3 slices and incubated for 30 min with 1-hydroxy-3-methoxycarbonyl-2, 2, 5, 5-tetramethylpyrrolidine HCl (CMH) in a thermo-regulated incubator (37 °C) under controlled pressure (20 mmHg) and gas mix  . EPR spectroscopy  was used to determine ROS production, expressed in µmol/min/mg dry weight.Immediately after harvesting, gastrocnemius muscles were placed in Krebs solution containing NaCl 99 mM, KCl 4.69 mM, CaClAll results were expressed as means ± SEM. Data were analysed using Prism software , and differences between groups were assessed using two-way ANOVA test, followed by the Bonferroni post-test.p value when considering ROS, paired t–test was performed. A p value < 0.05 was considered significant.To determine the precise 0  of ischemic limbs, as compared to contralateral non-ischemic limbs (7.58 ± 1.16 vs 2.63 ± 0.74 pmolO2/s/mg wet weight (ww), −62.8 ± 9.5%, p < 0.01, in non-ischemic and ischemic limbs, respectively, Two h ischemia followed by 2 h reperfusion significantly decreased Vmax, which corresponds to complexes I, III, IV, and V of the mitochondrial respiratory chain, decreased from 11.79 ± 1.74 to 4.65 ± 1.11 pmolO2/s/mg ww  decreased from 43.03 ± 4.55 to 35.85 ± 3.24 pmolO2/s/mg ww ; −61.1 ±6.8 % p = 0.08), as seen in Production of ROS was 0.08 ± 0.01 in the non-ischemic limbs and 0.13 ± 0.02 μmol/min/mg dw in the ischemic limbs  to 1.66 ± 0.44 μmol/mg dw (ischemic limbs), , as shown in Production of ROS in sildenafil-treated mice was 0.10 ± 0.01 in the non-ischemic limbs and 0.11 ± 0.01 μmol/min/mgdw in the ischemic limbs  failed to reach statistical significance.The main findings of this study are that lower limb IR impaired mitochondrial oxidative capacity and decreased calcium retention capacity in skeletal muscle. Further, albeit reducing ROS production, acute preconditioning with the phosphodiesterase 5 inhibitor sildenafil did not reduce IR-induced muscle injuries.Using different substrates, we observed that the functional activities of the main mitochondrial respiratory chain complexes were impaired by IR. The gastrocnemius muscle was chosen in view of its susceptibility to IR injuries ,36, alloSeveral studies previously reported a protective effect of sildenafil in IR settings. Thus, sildenafil protection was reported during acute cardiac IR in mouse , in the Accordingly, chronic administration of sildenafil improved ischemia-induced neovascularization in hypercholesterolemic apolipoprotein E-deficient mice, reduced TNF alpha staining in the femoral artery, and reduced inflammation and oxidative stress in skeletal muscle ,32,33.Nevertheless, our study shows that, although tending to decrease ROS production in line with literature data obtained both in skeletal and in cardiac muscles ,43, sildOne explanation might be that chronic treatment might be mandatory. Indeed, beneficial effects of sildenafil were observed after daily, weekly, or even longer administration ,32,33. HIn line with our results, Nio et al.  reportedThus, higher sildenafil doses might deserve further discussion. Higher dosages (40 and 10 mg/Kg) of oral sildenafil have been previously used ,33. HoweLower limb IR significantly impairs skeletal muscle mitochondrial functions. However, although beneficial in other IR settings and despite its tendency to decrease ROS production, acute pharmacologic preconditioning with the phosphodiesterase 5 inhibitor sildenafil did not reduce IR-induced impairments in mitochondrial oxidative and calcium retention capacities in skeletal muscle. Further studies will be useful to further determine the functional roles of PDE and cGMP and to investigate mitochondria-targeted antioxidants in normal and ischemic skeletal muscles .


Notably, aplysiatoxin induced similar levels of HIV-1 expression as prostratin but at up to 900-fold lower concentrations and without substantial effects on cell viability. Psammaplin A enhanced HIV-1 expression synergistically when treated in combination with the protein kinase C (PKC) activator prostratin, but not the histone deacetylase inhibitor (HDACi) panobinostat, suggesting that psammaplin A functions as a latency-reversing agent (LRA) of the HDACi class. Conversely, aplysiatoxin and debromoaplysiatoxin synergized with panobinostat but not prostratin, suggesting that they function as PKC activators. Our study identifies new compounds from previously untested marine natural products and adds to the repertoire of LRAs that can inform therapeutic “shock-and-kill”-based strategies to eliminate latent HIV-infected reservoirs.Natural products originating from marine and plant materials are a rich source of chemical diversity and unique antimicrobials. Using an established  As these proviruses can reactivate at any time to produce infectious virus, novel approaches are needed to eliminate these HIV-1 reservoirs ,3. One tefficacy ,6,7. Towefficacy ,13,14,1550s) of 3.8 µM or less, including at least one (bengamide A) that acts by mechanisms which are distinct from licensed antiretrovirals [Natural products obtained from plant and marine sources are a rich source of diverse chemical compounds, including HIV-1 inhibitors and novel LRAs ,17. Morerovirals . Here weAll natural compounds were confirmed ≥ 95% pure by NMR and LC/MS and dissolved in DMSO to stock concentrations of 5 mg/mL. The control LRA TNFα  was dissolved in PBS plus 0.1% bovine serum albumin to a stock concentration of 5 µg/mL. The control LRA panobinostat  was dissolved in DMSO to a stock concentration of 30 mM. The control LRA prostratin  was dissolved in PBS to a stock concentration of 1.5 mM.2.J-Lat Full Length T cell lines  were obtained from the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH (contributed by Dr. Eric Verdin) . These c6 cells/mL. 2 × 105 cells were then seeded into 96-well plate wells with compounds or control LRAs at desired final concentrations. Control cells were incubated at a final concentration of up to 0.3% DMSO vehicle control. In no case did final test concentrations of DMSO exceed 0.3%, which had no observable effects on cell viability or GFP expression. Cells were incubated at 37 °C and 5% CO2 for 24 h, after which a minimum of 5000 cells from each culture was analyzed by flow cytometry . Flow cytometry data were analyzed using FlowJo v. 8.8.7 software , and results were reported as the mean ± s.e.m. from at least 3 independent experiments.For latency-reversal assays, J-Lat cells were re-suspended in R10+ medium to a concentration of 1050s for panobinostat and prostratin were calculated to be 0.10 ± 0.02 and 7.1 ± 2.8 µM, respectively , parental Jurkat cells , which do not harbor integrated HIV-1 provirus, were prepared and treated with LRAs as described above. Following 24 h incubation, compound toxicity was assessed by measuring surface expression of the early apoptotic marker annexin V by flow cytometry . Results were reported as the fold-increase in annexin V-positive cells relative to 0.1% DMSO-treated control cells (mean ± s.e.m.) from at least 3 independent experiments. Representative data are shown in We next screened 257 structurally-diverse pure compounds derived from marine natural products at 2.5 µg/mL for latency reversal activity in J-Lat 9.2 cells. Of these, seven (2.7%) compounds resulted in cytolysis and disruption of cell morphology as observed by light microscopy, consistent with widespread cell death, and were not considered further. Of the remaining 250 compounds, four induced GFP expression in at least 4% of cells A. This “Poecillastra sp. and Jaspis sp. [Phorbas sp., is a potent activator of cyclic AMP and PKC signaling that we recently characterized for its HIV-1 latency reversal activity [Structures of the four identified compounds are shown in spis sp. , was prespis sp. ,31. In cspis sp. ,33,34. Hspis sp. ,37,38, aactivity ,20 and i50 was calculated as 1.9 ± 0.3 µM, approximately 19-fold higher than the EC50 of panobinostat  and yielded a calculated EC50 of 0.92 ± 0.14 µM, or 7.7-fold more potent than prostratin.Each new LRA induced dose-dependent reversal of HIV-1 latency in J-Lat cells across multiple concentrations C,D. The binostat . In contIn Jurkat cells, we observed that 3.8 µM psammaplin A induced a 3.3 ± 0.6-fold increase in annexin V staining, with extensive cell death observed at higher concentrations. In contrast, no more than 2.0 ± 0.2 and 1.6 ± 0.1-fold increases in annexin V staining were observed for aplysiatoxin and debromoaplysiatoxin, respectively E,F. Thus50 of psammaplin A was calculated as 1.5 ± 0.1 µM, or approximately 20.5-fold higher than the EC50 of panobinostat  and a calculated EC50 of 0.0033 ± 0.0012 µM, or 540-fold more potent than prostratin and 12.4-fold more than panobinostat. Finally, the calculated EC50 of debromoaplysiatoxin (0.081 ± 0.029 µM) was 22.2-fold more potent than prostratin. Thus, the rank-order of potency for all LRAs was consistent across all cell lines.In J-Lat 10.6 cells, we observed that all LRAs were capable of inducing GFP expression in at least two-thirds of cells B, indicabinostat . MoreoveGag antibody KC57-RD1  and processed using the Cytofix/Cytoperm Fixation/Permeabilization Kit  prior to flow cytometric analysis. Results were then reported as the fold-increase in p24Gag-positive cells relative to 0.1% DMSO-treated control cells (mean ± s.e.m.) from at least 3 independent experiments. All LRAs were observed to induce at least 9.7-fold increased p24Gag-positive cells, with the same rank order as observed for GFP expression  plus ionomycin (1 µg/mL), 3.8 µM psammaplin A, 1.1 µM aplysiatoxin, 1.3 µM debromoaplysiatoxin, or 0.1% DMSO vehicle control. All conditions were performed in duplicate. Following 24 h incubation at 37 °C and 5% CO2, supernatant p24Gag protein was quantified by ELISA , and cell viability was measured by flow cytometry using Guava Viacount, a DNA intercalating dye (EMD Millipore). In most cases, each LRA caused an increase in supernatant p24Gag above background; however, substantial donor-to-donor variation was observed, consistent with other studies [Gag relative to untreated cells, psammaplin A resulted in a 85.0 ± 41.6% increase. Similarly, aplysiatoxin and debromoaplysiatoxin induced increases of 56.0 ± 15.0 and 46.9 ± 22.3%, respectively. No major changes in cell viability were observed, with a maximum 15.4 ± 2.2% (mean ± s.e.m.) reduction in viability in the presence of 3.8 µM psammaplin A  from three HIV-infected donors on stably-suppressive antiretroviral therapy for at least three years . Study p studies ,12  as measured by the Bliss independence model, which was calculated as described previously [As described previously ,13,14,15eviously ,11.Treatment of J-Lat 9.2 cells with 2 µM psammaplin A in addition to TNFα or prostratin induced 49.8 ± 1.1 and 44.4 ± 5.1% GFP-positive cells, representing 1.7- and 2.7-fold increases in GFP expression over what would be expected from strictly additive effects, respectively. However, treatment of J-Lat cells with psammaplin A plus panobinostat induced only 9.8 ± 1.7% GFP-positive cells, or 0.6-fold of what would be expected by strictly additive effects . These rIn summary, we identify four LRAs derived from marine natural products that can be added to the repertoire of known HIV-1 shock-and-kill agents. The likely mechanisms of action for all four compounds, supported here and by prior studies, are consistent with established functional classes, including one HDACi (psammaplin A) and three PKC activators  . Dose reOur previous discovery of multiple novel HIV inhibitors from this chemically-diverse library  led us Taken together, this study highlights the benefits of natural product-based screens for LRA discovery. The identification and evaluation of new LRAs will support the development of novel therapeutic combinations and clinical approaches to reduce or eliminate latent HIV-1 reservoirs.


Background: NLRP3 inflammasome activation in response to several signals, including mitochondrial DNA (mDNA), regulates inflammatory responses by caspase-1 activation and interleukin-1β (IL-1β) release. Circulating mDNA is linked to micro and macrovascular complications in diabetes. However, a role for mDNA in endothelial dysfunction is not clear. We tested the hypothesis that mDNA contributes to diabetes-associated endothelial dysfunction and vascular inflammation via NLRP3 activation.Methods: Vascular reactivity, reactive oxygen species (ROS) generation, calcium (Ca2+) influx and caspase-1 and IL-1β activation were determined in mesenteric resistance arteries from normoglicemic and streptozotocin-induced diabetic C57BL/6 and NLRP3 knockout (–/–Nlrp3) mice. Endothelial cells and mesenteric arteries were stimulated with mDNA from control (cmDNA) and diabetic (dmDNA) mice.Results: Diabetes reduced endothelium-dependent vasodilation and increased vascular ROS generation and caspase-1 and IL-1β activation in C57BL/6, but not in –/–Nlrp3 mice. Diabetes increased pancreatic cytosolic mDNA. dmDNA decreased endothelium-dependent vasodilation. In endothelial cells, dmDNA activated NLRP3 via mitochondrial ROS and Ca2+ influx. Patients with type 1 diabetes exhibited increased circulating mDNA as well as caspase-1 and IL-1β activation.Conclusion: dmDNA activates endothelial NLRP3 inflammasome by mechanisms that involve Ca2+ influx and mitochondrial ROS generation. NLRP3 deficiency prevents diabetes-associated vascular inflammatory damage and endothelial dysfunction. Our study highlights the importance of NLRP3 inflammasome in diabetes-associated vascular dysfunction, which is key to diabetic complications. The NLRP3 inflammasome is a multi-protein complex present in cells of the adaptive and innate immune system. NLRP3 regulates inflammatory responses by oligomerization and recruitment of the apoptosis-associated speck-like protein containing a caspase recruit domain (ASC) and pro-caspase-1, causing auto-cleavage and activation of caspase-1, which, in turn, cleaves pro-IL-1β and pro-IL-18 into mature cytokines . Mature +) efflux, calcium (Ca2+) influx, and DAMPs (damage-associated molecular patterns) also induce NLRP3 oligomerization (Increased availability of reactive oxygen species (ROS) is a very important mechanism that triggers endothelial dysfunction, being involved in the onset of many diseases. Increased ROS is also a classical mechanism that induces NLRP3 inflammasome activation. Many other stimuli, such as lysosomal damage, potassium  and approved by the Ethics Committee on Animal Research of the Ribeirão Preto Medical School – University of São Paulo, Ribeirão Preto, Brazil (protocol no. 26/2015).All experimental protocols were performed in accordance with the ARRIVE Guidelines  mice were obtained from the Isogenic Breeding Unit of the Ribeirão Preto Medical School, University of São Paulo, Ribeirão Preto, Brazil. The animals were housed in the animal facility of the Pharmacology Department, Ribeirão Preto Medical School, on 12-h light/dark cycles under controlled temperature (22 ± 1°C) with ad libitum access to food and water. After a 1-week acclimatization period, mice were randomly divided into non-diabetic and diabetic groups.Male, 8 to 10 week-old C57BL/6 wild-type (WT) and NLRP3 receptor knockout  dissolved in 0.1 M sodium citrate (pH 4.5) for five consecutive days. Blood glucose levels, body weight, and diabetes incidence were monitored weekly. Mice were considered diabetic when glucose levels were ≥230 mg/dl after two consecutive determinations under non-fasting conditions. The animals were submitted to experimental protocols 30 days after induction of diabetes. Body weight, blood glucose, and insulin levels are shown in Mice were given daily intraperitoneal injections of 40 mg/kg of streptozotocin . Finally, pancreatic mDNA isolated from control (cmDNA) and diabetic (dmDNA) mice was quantified using an EpochTM Microplate apparatus .Pancreata from non-diabetic and diabetic mice were submitted to protocols for mitochondria isolation. The pancreatic tissue was homogenized in 5 ml of medium  at pH 7.2, centrifuged at 600 2) chamber. Segments of second-branch mesenteric arteries (2 mm in length) were mounted in a small vessel myograph . Arteries were maintained in Krebs Henseleit solution , at 37°C, pH 7.4, and gassed with a mixture of 95% O2 and 5% CO2.The method described by + (120 mM)] to evaluate the contractile capacity. After washing and return to the basal tension, arteries were contracted with phenylephrine (10–6 M) and stimulated with acetylcholine (10–5 M) to determine the presence of a functional endothelium. Arteries exhibiting a vasodilator response to acetylcholine greater than 80% were considered endothelium-intact vessels. The failure of acetylcholine to elicit relaxation of arteries that were subjected to rubbing of the intimal surface was taken as proof of endothelium removal. After washing and another period of stabilization, concentration-response curves to acetylcholine and sodium nitroprusside were performed.Mesenteric arteries preparations were set to reach a tension of 13.3 kPa  and remained at rest for 30 min for stabilization. The arteries were stimulated with Krebs solution containing a high concentration of potassium  and 5 μl of lucigenin (5 μM) for basal luminescence reading. Then, 50 μl of NAD(P)H (100 μM) were added to the tube and superoxide anion generation was quantified using the Line TL Tube Luminometer . Superoxide anion generation was expressed in RLU (relative units of luminescence)/dry weight (g).Superoxide anion generation was determined in mice mesenteric arteries and 2PO4 20, EGTA 1] containing a protease inhibitor cocktail , and then transferred to Eppendorf tubes. In a 96-well white plate, 50 μl of sample, 173.75 μl of phosphate buffer  and 1.25 μl of lucigenin were pipetted in each well. Basal luminescence reading (3 min) was performed, and 25 μl of NAD(P)H (1 mM) was then added and a new reading (after 15 min) was performed. The Orion II Microplate Luminometer  was used. Superoxide anion generation was expressed in RLU/μg protein.mDNA-stimulated endothelial cells were mechanically removed with 100 μl lysis buffer  and Rmax , respectively.Results are expressed as mean ±standard error of the mean (E.P.M.). Relaxation responses are expressed as the percentage of relaxation in relation to pre-contraction levels induced by phenylephrine. Concentration-effect curves were submitted to non-linear regression analysis using the GraphPad Prism 6.0 program , T1D = 3.8 ± 0.1 vs. control = 0.9 ± 0.2;  < 0.05] , and cas < 0.05]  as well  < 0.05]  were incor IL-1β .2, T1D = 6.2 ± 0.2 vs. control = 6.6 ± 0.1; Rmax, T1D = 47.2 ± 4.9 vs. control = 90.2 ± 0.1; P < 0.05)        (Endothelium-denuded arteries from control and T1D mice exhibited similar relaxation responses to sodium nitroprusside . Mesente < 0.05) . NLRP3 d < 0.05) . Reduced < 0.05) , and com < 0.05)  and by p < 0.05) . MCC950  > 0.05) , whereas > 0.05)  and Peg- > 0.05)  producedP < 0.05) (P < 0.05)   as well  < 0.05)  in mesenP < 0.05), which was not observed in arteries from Nlrp3–/– mice   .P < 0.05) (P < 0.05) (P < 0.05) (P < 0.05)   in mesen < 0.05)  and by C < 0.05) . dmDNA a < 0.05)  and CCCP < 0.05) . Tiron aP < 0.05) (P < 0.05) (P < 0.05) (P > 0.05).The incubation of endothelial cells with cmDNA and dmDNA did not activate caspase-1 or IL-1β. However, in cells primed with LPS for 24 h prior to mDNA incubation, dmDNA, but not cmDNA, increased activation of caspase-1   and IL-1 < 0.05) . MCC950  < 0.05) . MCC950 2+ influx   (P < 0.05) in endothelial cells  . The pre < 0.05)  and caspal cells .P < 0.05) (P < 0.05) (P < 0.05)  , caspase < 0.05)  and IL-1 < 0.05) . Clinica < 0.05) .Diabetes did not change 8-OHdG levels in serum DNA or mouse pancreatic mDNA .Nlrp3–/– mice are protected from diabetes-associated inflammatory vascular damage and endothelial dysfunction. Mitochondrial DNA is key for NLRP3 inflammasome activation in endothelial cells through increased Ca2+ influx and mitochondrial ROS generation.The present study shows that In vitro experiments also show a modulatory role of NLRP3 on endothelial function since the silencing of NLRP3 gene prevents caspase-1 and IL-1β activation in endothelial cells stimulated with cell wall fragments of Lactobacillus casei function has been previously suggested in other studies (15–17). In obese rats, vascular dysfunction, represented by impaired endothelium-dependent relaxation, is associated with increased vascular expression of inflammasome markers such as NLRP3, caspase-1 and IL-1β . Mice wius casei . Furtherus casei .Reactive oxygen species modulate endothelial function and IL-1β is an important stimulus for ROS production. IL-1β increases ROS generation in human umbilical vein endothelial cells (HUVECs)  and in vNlrp3–/– mice with type 1 diabetes displayed decreased expression of Nox4 and reduced ROS levels. Other studies support an interaction between NLRP3 inflammasome and Nox expression and activity. Nox4 inhibition prevents ROS generation and decreases protein expression of NLRP3, caspase-1 and IL-1β in endothelial cells stimulated with high glucose  in plasmocytoid dendritic cells . One of Another potential mechanism by which mDNA may activate the NLRP3 inflammasome is mitochondrial ROS generation. Human macrophages incubated with increasing doses of inhibitors of the mitochondrial respiratory chain display increased IL-1β release. This effect is inhibited by NLRP3 and caspase-1 gene deletion in both human and murine macrophages . In our 2+ influx is also critical for NLRP3 activation and, consequently, IL-1β release. In LPS-primed and ATP-stimulated macrophages, IL-1β release is prevented by thapsigargin, a Ca2+ mobilization agent that inhibits the sarcoplasmic Ca2+-ATPase . The patients/participants provided their written informed consent to participate in this study. The animal study was reviewed and approved by the Ethics Committee on Animal Research of the Ribeirão Preto Medical School – University of São Paulo, Ribeirão Preto, Brazil (protocol no. 26/2015).CP, NF, CZ, and JFS performed the wet laboratory experiments. CP, DC, and RT designed the study. VG and DV provided the human samples. DZ and JSS provided the knockout mice used in this study. CP wrote the manuscript. DC, DZ, and RT revised its scientific content.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.


Helicotylenchus dihystera (Hd) and Scutellonema brachyurus (Sb) appear as potential pathogens for soybean in Brazil, since their spread and population densities have been increased on soybean growing areas. Aiming to evaluate the quantitative distribution of Hd and Sb in soybean fields in South Brazil, a survey was conducted during the growing seasons of 2014–2015 and 2015–2016 in which 1,088 soil samples and 1,043 root samples were analyzed. Besides, two greenhouse experiments were conducted to evaluate the pathogenicity of both nematodes to soybean plants, in comparison with P. brachyurus (Pb), a known pathogen of the crop. The survey demonstrated that Pb, Hd and Sb are widely distributed in the States of Paraná, Santa Catarina and Mato Grosso do Sul. Besides, we proved that Hd and Sb multiply and cause root lesions in soybean cv. Potência, since they were found inside roots, and can be considered as potential pathogens for soybean plants.Nematode diseases have a worldwide importance for several economic agricultural crops, such as soybean. Frequently, new or secondary pathogens arise as emergent diseases due to the intensified use of agricultural lands, causing extensive yield losses.  The expansion of problems caused by nematodes in Brazil, especially in the Cerrado region, probably is due to the intensified use of agricultural lands, with monoculture of susceptible crops, which increase soil nematode populations along the crops. The economy in this region is based largely on the viability of soybean, maize, and cotton .Meloidogyne spp. Goeldi 1929) and the soybean cyst nematode (SCN), Heterodera glycines Ichinoe, represent the most economically important nematodes for soybean crop in Brazil and worldwide [H. glycines, two important changes occurred, the use of alternative plants for crop rotation and the increase in the no-tillage system [Pratylenchus brachyurus (Godfrey 1929) Filipjev & Sch. Stekhoven 1974 populations, which currently is a concern as one of the main problems to soybean in Brazilian fields [Root-knot nematodes (RKNs)  [On soybean, yield losses caused by 0% clay) .Helicotylenchus dihystera (Cobb) Sher and Scutellonema brachyurus Andrassy, appear as potential pathogens for soybean in Brazil, since their spread and population densities have been increased on soybean growing areas [Recently, other species, as ng areas , 8. The ng areas .Scutellonema brachyurus has been observed especially in the States of Paraná, Mato Grosso do Sul and Maranhão, in soybean fields showing stunting plants associated with yield losses [3 of soil in samples collected in soybean fields. Apparently, injuries are associated with drought and the use of early cultivars [d losses . Soil neultivars .Helicotylenchus dihystera, mainly associated with crop losses on sugarcane [H. dihystera was found in 85%, 92%, and 47% of the samples, respectively [ugarcane  and maizugarcane , has alsugarcane . Surveysectively , 12. GenH. dihystera in soybean plants was already reported by Orbin [H. dihystera and observed adults, larvae and eggs within the root cortex associated with small brown lesions in the immediate vicinity of the nematodes. However, because of the lack of persistent burrows, the author suggested that H. dihystera was not a significant pathogen of soybean.The parasitism of by Orbin , that stH. dihystera and S. brachyurus in soybean fields from the States of Paraná, Mato Grosso do Sul and Santa Catarina, a survey was conducted during the growing seasons of 2014–2015 and 2015–2016 in which 1,088 soil samples and 1,043 root samples were analyzed. Besides, two greenhouse experiments were conducted to evaluate the pathogenicity of both nematodes on soybean plants, in comparison with P. brachyurus, a known pathogen of the crop.In order to evaluate the quantitative distribution of Surveys were undertaken in commercial soybean fields in the States of Paraná , Mato Grosso do Sul  and Santa Catarina , in the Southern Brazil , between3 subsample using the Baermann funnel method [Pratylenchus brachyurus (Pb), Helicotylenchus dihystera (Hd) and Scutellonema brachyurus (Sb) were identified through morphological and morphometrical approaches, based on measurements of 10 adult females from each population [The entire root system was washed free of soil, weighed and used to extract nematodes by the maceration procedure described in literature  without l method . Nematodpulation , 16, 17.Characters measured were: body length (L), stylet length (St), height (Hstk) and width (Wstk) of stylet knobs, distance from posterior end to vulva (V), distance from vulva to anus (V-A), tail length (T), post-uterine branch length (PUB), dorsal esophageal gland orifice (DGO), maximum body diameter (BD), body diameter at anus (BDA) and vulva (BDV) to Pb; L, V, lip height (LH), lip diameter (LD), St, conus length (CL), DGO, pharynx (P), excretory pore (EP), BD, T, and BDA to Hd; and L, V, DGO, St, metenchium length (ML), telenchium length (TL), P, EP, pharyngeal overlap (PO), BD, BDA, medium bulb length (MBL) and diameter (MBD), LH, LD, lateral field width (LFW), T, scutellum length (SCL) and width (SCW), and spermatheca length (SPL) and width (SPW) to Sb.5’- TTG ATT ACG TCC CTG CCC TTT -3’) and RN58SR (5’- ACG AGC CGA GTG ATC CAC CG -3’) to ITS-1 region, located respectively in the 18S and 5.8S rDNA [TCGGAAGGAACCAGCTACTA) and D3B (ACAAGTACCGTGAGGGAAAGTTG) to D2/D3 region.Besides, species delimitation was undertaken using an integrated approach considering morphological and morphometric evaluation combined with molecular sequence analyses . For mol.8S rDNA , and D2AFor PCR, 21 μl of the master mix Platinum PCR Supermix (Invitrogen) were added to a 0.2 ml microcentrifuge tube; after, 2 μl of DNA and 1 μl of each primer were added. Amplifications conditions for ITS-1 were as follows: 94 °C for 2 min 45 s followed by 40 cycles of 94 °C for 1 min, 57 °C for 45 s and 72 °C for 2 min, and a final extension of 72 °C for 10 min; for D2/D3: 94 °C for 5 min followed by 35 cycles of 94 °C for 30 s, 55 °C for 45 s and 72 °C for 2 min, and a final extension of 72 °C for 10 min. Purified DNA fragments were sequenced on a sequencer ABI 377 DNA Sequencer and the quality of sequences was checked using the software BioEdit. Detailed protocols were described by our colleagues  (Hd) andTwo greenhouse experiments were carried out at the Instituto Agronômico do Paraná, Londrina, Paraná State, Brazil . The trials were conducted from 11 November 2016 to 13 January 2017 with the temperature ranging from 22 °C to 41 °C.3 plastic pots containing 400 cm3 of sterilized (160°C/5hours) soil. Seedlings were thinned to one per pot prior to nematode inoculation. Pratylenchus brachyurus population used as inoculum was collected in São Luís, Maranhão State, Brazil , from soybean plants (Glycine max), identified through morphological [Helicotylenchus dihystera and Scutellonema brachyurus populations were collected in Fênix, Paraná State, Brazil , both from soybean plants, identified through morphological and molecular approaches [Seeds of soybean cultivar BMX Potência were sown in 500 cmological  techniquproaches  and subs® Plus .After nematode extraction from soybean roots by the maceration procedure described in literature , withoutThe final nematode population (FP) was evaluated 15, 30 and 70 days after inoculation (DAI). At 15 and 30 DAI, plants were removed from pots and root systems were washed carefully and stained with fuchsin acid . The entExperiments were arranged in completely randomized design, with treatments corresponding to each nematode species and 8 replicates. Data were submitted to variance analysis and, for data residuals normalization, BoxCox procedure indicated that data transformation by ln(y+0.01) was necessary. Treatments were compared by Fisher´s Least Significant Difference (LSD) Test (P ≤ 0.05), using the R 2.15.2 program , package3 of soil; for Hd, densities ranged from 4 to 168 Nema/g and from 1 to 4,284 nematodes per 50 cm3 of soil, whereas to Sb densities ranged from 3 to 379 Nema/g and from 1 to 11,712 nematodes per 50 cm3 of soil. Generally, growers in these localities described symptoms of stunting and chlorotic plants, associated with lower productivities, but no quantifications were done, since other nematode species occurred in the samples (Pb was found in 501 root samples (48.03%) and in 568 soil samples (52.21%), Hd in 300 root (28.76%) and in 843 soil (77.48%) samples, and Sb in 164 root (15.72%) and in 328 soil (30.15%) samples . The pop samples  and the P. brachyurus values measured, in μm (mean ± standard deviation), were: L = 543 (± 19.50), BD = 24 (± 2.20), BDV = 21 (± 1.43), BDA = 15 (± 1.24), V% = 82 (± 4.55), St = 19 (± 0.81), Wstk = 4 (± 0.29), Hstk = 3 (± 0.44), DGO = 2.78 (± 0.10), V-A = 54 (± 4.37), T = 28 (± 1.52), and PUB = 16 (± 2.41). For H. dihystera females, values measured, in μm (mean ± standard deviation), were:: L = 601 (± 44.30), V = 209 (± 23.58), LH = 4 (± 0.32), LD = 7 (± 0.48), St = 26 (± 1.01), CL = 12 (± 0.86), DGO = 13 (± 1.01), P = 142 (± 8.30), EP = 108 (± 6.94), BD = 25 (± 1.83), T = 16 (± 1.63), and BDA = 16 (± 1.27). Finally, for S. brachyurus females, values measured, in μm (mean ± standard deviation), were:: L = 740 (± 30.06), V = 285 (± 18.67), DGO = 8 (± 0.62), St = 29 (± 1.23), ML = 14 (± 1.17), TL = 12 (± 1.11), P = 149 (± 8.03), EP = 116 (± 9.32), PO = 23 (± 2.39), BD = 29 (± 1.27), BDA = 19 (± 0.72), MBL = 12 (± 0.78), MBD = 10 (± 0.56), LH = 6 (± 0.26), LD = 9 (± 0.36), LFW = 4 (± 0.48), T = 4 (± 1.01), SCL = 5 (± 0.49), SCW = 4 (± 0.58), SPL = 11 (± 1.09), and SPW = 10 (± 0.92).Except for slightly differences in tail shape, morphology of specimens of Pb, Hd and Sb  compare At 15 and 30 DAI, the observation of roots stained with fuchsin acid showed that the three nematode species penetrated soybean roots cv. Potência Figs  and 5. IH. dihystera and S. brachyurus, in relation to P. brachyurus, at 15 DAI in soil, in both experiments  but can also feed on fungi [Aphelenchoides spp. in adaptation to hosts imposed in the cropping systems. The same plasticity could be suggested to exist in Hd and Sb.Both nematodes could facultatively had developed the migratory endoparasitic habit in order to better adaptation in hosts used in a cropping system, as the intensified use of agricultural lands provide roots along the year for nematode parasitism and adaptation. As an example, the evolution of plant parasitism in nematodes is reported for on fungi . Recentlon fungi , and alson fungi , which sSince Hd and Sb were found inside soybean roots, root lesions and generalized darkening of root systems were observed, as a result of this type of parasitism (as showed in S1 File(PDF)Click here for additional data file.S2 File(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(PDF)Click here for additional data file.S5 File(PDF)Click here for additional data file.S6 File(PDF)Click here for additional data file.S7 File(PDF)Click here for additional data file.S8 File(PDF)Click here for additional data file.


Peumus boldus), which is used around the world as a traditional herbal medicinal product (THMP), mostly to relieve gastrointestinal disorders. This biomass may be a resource for the agrochemical industry to manufacture botanical insecticides. In this regard, the insecticidal potential of boldo has been poorly investigated. In the present work, hydrodistillation of a commercial boldo folium gave 1.5% (w/w) of a yellowish essential oil  containing 1,8-cineole (20.7%), p-cymene (18.5%), limonene (9.1%), ascaridole (9.1%) and β-phellandrene (6.4%) as the main constituents, as determined by gas chromatography-mass spectrometry (GC-MS). NMR analysis allowed us to determine that ascaridole was mainly represented by the cis-isomer. BEO was toxic to larvae of the filariasis vector Culex quinquefasciatus and adults of the housefly Musca domestica, showing LC50/LD50 values of 67.9 mg·L−1 and 98.5 µg·adult−1, respectively. On the other hand, lower insecticidal activity was observed against larvae of the moth pest Spodoptera littoralis (LD50 of 268.9 µg·larva−1). It is worth noting that, when tested at LC90 concentration, BEO was significantly less toxic to aquatic microcrustacean Daphnia magna than the conventional insecticide α-cypermethrin. Finally, in the attempt to explore the BEO mode of action, we tested it for acetylcholinesterase (AChE) inhibitory properties using the Ellman method, obtaining negligible effects (IC50 = 0.45 mg·mL−1). Taken together, these results gave new insights into the potential of BEO as a future ingredient of botanical insecticides.Every year Chile exports about 2000 tons of boldo folium ( Peumus boldus Molina) is an evergreen tree belonging to the Monimiaceae family and native to temperate regions of Chile. It is a dioecious, small tree, up to 6 m tall, having an olive-like habitus, occurring as a single plant or in forests between the flat areas and the coastal cordilleras +. Anal. calcd. for (C10H16O2) C, 71.39; H, 9.59; Found: C, 71.38; H, 9.61.Cx. quinquefasciatus. Mosquito larvicidal assays were carried out according to WHO Standard Procedures, with slight modifications [−1) was added to 224 mL of distilled water in a 500-mL glass bowl and shaken lightly to ensure a homogenous test solution. The selected larvae  were transferred in water into a bowl of prepared test solution with a final surface area of 125 cm−2. Distilled water containing the same amount of DMSO used to test EO was used as negative control and α-cypermethrin (Vaztak®) was used as the positive control . After 24 h treatment mortality was determined. Laboratory conditions were 25 ± 1 °C, 70 ± 3% R.H. and 16:8 h (L:D) during both rearing and toxicity tests.The acute toxicity, measured as mortality after 24 h of exposure, was determined on 3rd instar larvae of ications . For expM. domestica adult females (2–5 days old) [2, were treated with 1 μL of acetone  containing BEO at doses of 30, 50, 100, 130, 160, 180 and 200 μg·adult−1  using a microelectric applicator. Acetone was the negative control and α-cypermethrin (Vaztak®) at 0.05, 0.1, 0.2, 0.3, 0.5, 0.7, 0.9 and 1.2 μg·adult−1 was used as the positive control. Post-treatment, females were transferred to a recovery box (10 × 10 × 12 cm) for 24 h and mortality was determined. Laboratory conditions were 25 ± 1 °C, 70 ± 3% R.H. and 16:8 h (L:D) during both rearing and toxicity tests.The acute toxicity, measured as mortality after 24 h of exposure, was determined by topical application on ays old) . HouseflS. littoralis (3rd instar) following our earlier method [−1. Four groups, each composed of 20 larvae, were tested for each dose. Acetone was the negative control and α-cypermethrin (Vaztak®) at 0.005, 0.008, 0.01, 0.015 and 0.02 μg·larva−1 was used as the positive control. After treatment, larvae were transferred to a recovery box (10 × 10 × 7 cm) for 24 h for mortality determination. Laboratory conditions were 25 ± 1 °C, 70 ± 3% R.H. and 16:8 h (L:D) during both rearing and toxicity tests. Death was recorded when the larvae did not respond to prodding with forceps.The acute toxicity of BEO and α-cypermethrin, measured as mortality after 24 h of exposure, was determined by topical application on larvae (weight ranging from 20 to 25 mg) of r method . Boldo ED. magna adults (2–5 days old) were used as a non-target organisms to assess the impact of BEO on aquatic invertebrates, also testing α-cypermethrin as positive control. Twenty D. magna were exposed in each test vessel and four replicates were carried out, for a total of 80 D. magna per treatment. D. magna individuals were placed in containers with 250 mL of pure water, and BEO or α-cypermethrin was then mixed into the water at concentrations corresponding to the LC90 estimated on Cx. quinquefasciatus larvae (for EO = 96 mg·L−1 and for α-cypermethrin = 0.025 mg·L−1). The chemicals were emulsified using DMSO (in the ratio 1:1). Mortality was calculated after 24 and 48 h. Laboratory conditions were 25 ± 1 °C, 70 ± 3% R.H. and 16:8 h (L:D) during both rearing and toxicity tests.The acute toxicity tests were carried out in accordance with the guidelines of the OECD with slight modifications ,59. LaboInhibition of AChE activity of BEO was measured following the method of Ellmann with slight modifications . Briefly50(90) or LC50(90) for the targeted organisms, with associated 95% CL and chi-square values, were calculated using probit analysis [50) was calculated via nonlinear regression analysis.When control mortality did not exceed 20%, the Abbott’s formula  was applanalysis . The conTaken together, our findings indicate that the analyzed BEO can be considered a relatively safe pest and vector control product, due to the low content of ascaridole (toxic to vertebrates), thus presenting a potential use in the area of botanical insecticide development. Starting from the present data showing low inhibition of AChE triggered by BEO, further studies are necessary to shed light on its modes of action, with special reference to the potential inhibition of GABA-gated chloride channels and/or binding to octopamine receptors, as well as to assess its efficacy on other target insects of agricultural and public health importance. Besides, its safety towards other beneficial organisms still needs to be elucidated in real-world conditions, to ascertain the eco-friendliness of this natural product and its potential as ingredient in botanical insecticides. Its scalability on an industrial level may be assured by the huge biomass of boldo folium, which is exported all around the world and used to make pharmaceutics and herbal remedies.


An enhanced cellular accumulation of fluorescently labeled CNP°-CGA in the human renal cancer cells (786-O) as early as 30 min and increased time-dependently were observed through fluorescent microscopic visualization and flow cytometric assessment. A significant concentration-dependent antiproliferation activity of encapsulated CGA was achieved at IC50 of 16.20 µM as compared to CGA itself (unable to determine from the cell proliferative assay), implying that the competent delivery vector, chitosan nanoparticle, is able to enhance the intracellular accumulation, antiproliferative activity, and antioxidant properties of CGA at lower concentration as compared to CGA alone.Naturally existing Chlorogenic acid (CGA) is an antioxidant-rich compound reported to act a chemopreventive agent by scavenging free radicals and suppressing cancer-causing mechanisms. Conversely, the compound’s poor thermal and pH  stability, poor solubility, and low cellular permeability have been a huge hindrance for it to exhibit its efficacy as a nutraceutical compound. Supposedly, encapsulation of CGA in chitosan nanoparticles (CNP), nano-sized colloidal delivery vector, could possibly assist in enhancing its antioxidant properties, in vitro cellular accumulation, and increase chemopreventive efficacy at a lower concentration. Hence, in this study, a stable, monodispersed, non-toxic CNP synthesized via ionic gelation method at an optimum parameter (600 µL of 0.5 mg/mL of chitosan and 200 µL of 0.7 mg/mL of tripolyphosphate), denoted as CNP°, was used to encapsulate CGA. Sequence of physicochemical analyses and morphological studies were performed to discern the successful formation of the CNP°-CGA hybrid. Antioxidant property  assay), in vitro antiproliferative activity of CNP°-CGA, and in vitro accumulation of fluorescently labeled (FITC) CNP°-CGA in cancer cells were evaluated. Findings revealed that successful formation of CNP°-CGA hybrid was reveled through an increase in particle size 134.44 ± 18.29 nm (polydispersity index (PDI) 0.29 ± 0.03) as compared to empty CNP°, 80.89 ± 5.16 nm (PDI 0.26 ± 0.01) with a maximal of 12.04 μM CGA loaded per unit weight of CNP° using 20 µM of CGA. This result correlated with Fourier-Transform Infrared (FTIR) spectroscopic analysis, transmission Electron Microscopy (TEM) and field emission scanning (FESEM) electron microscopy, and ImageJ evaluation. The scavenging activity of CNP°-CGA (IC Lonicera japonica Thunb. [Artemisia capillaris, Ilex paraguariensis, Pimpinella anisum, Cymbopogon citratus) [The history of therapeutic compounds discovery from various sources  can be traced back to the 1800s. One such compound include the naturally produced 5-O-caffeoylquinic acid (5-CQA) compound or commonly known as chlorogenic acid (CGA); an ester of caffeic acid and L-quinic acid . This pha Thunb. . The comitratus) ,5,6,7,8.CGA is an extensively studied plant secondary metabolite with potent antioxidant strength and other conceivable therapeutic properties. The presence of a catechol structure serves as the primary binding site for free radicals ,10. CGA Therewithal, studies have also shown potential participation of CGA as a nutraceutical compound in both in vitro and in vivo applications; which includes prevention against microorganisms ,16,17, aThe compound integrity of CGA is influenced by temperature and pH. CGA was reported to undergo isomerization or possibly other chemical reactions  under the exposure of high temperature (time-dependent), and at basic and neutral pH ,25,26,27Hence to confront these limitations, administration of a high dosage was used  but in w3+ of chitosan (CS) and anionic phosphate groups, -PO43− of cross-linker tripolyphosphate (TPP) [Alternatively, nanoparticles incorporated drug delivery systems (nano-DDS) have been proposed as an auxiliary avenue to deliver CGA to the targeted sites in controlled released mechanism with conserved antioxidant and therapeutic strength, minimized administration dosage, and lowered side effects ,37,38,39te (TPP) ,41. Due te (TPP) ,43,44.Collectively, this vector is able to protect the cargo from degradation by phagocytosis and circumvent drug resistance mechanisms, deliver therapeutic drugs to the targeted sites, provide an alternative route for insoluble drugs to permeate cells, improve the bioavailability and therapeutic efficacy of drugs in the living system, upturn the enhanced permeability and retention (EPR) effect, participate in sustained released rates of the drugs, and conserve the pharmacodynamics effects and in vivo stability of drugs ,47,48,49fCNP). Hence, validating the encapsulated CGA has high potential as a drug delivery vector in pharmaceutical applications.This study was outlined based on the objective of enhancing the in vitro accumulation and cytotoxicity effect on human kidney cancer cell line (786-O), and improvise the antioxidant activity of chlorogenic acid by encapsulating into fabricated stable, monodispersed, colloidal CNP as a delivery vector. The physicochemical characterizations, antioxidant strength, and in vitro cytotoxicity activities of CGA and CNP encapsulated CGA hybrid were analyzed and optimized for better intracellular efficacy of CGA. Additionally, the in vitro accumulation of CGA was studied using fluorescently labeled CNP (3+), particle size distributions, and the stability of the nanoparticles depicted in 2) of CS were protonated [43−) of TPP to form hydrophilic CNP. However, not all -NH3+ groups participated in the cross-linking reaction. Shown in 3+ groups and subsequently confirms the formation of nanoparticles. On an overall basis, the percentage of utilized -NH3+ in formation of nanoparticles increased with the TPP volumes.CNP were formulated using three parameters as described in otonated  spontane3+ graph showed an increase of 13.50% utilized -NH3+ (from 5.75 ± 0.82% to 19.25 ± 1.36%) as the TPP volume increased from 50 µL to 300 µL in CNP-F1. Likewise, the utilized -NH3+ percentage in CNP-F2 and CNP-F3 increased 9.12% (from 4.83 ± 0.89% to 13.95 ± 3.60%) and 9.01% (from 2.96 ± 1.29% to 11.97 ± 2.34%), respectively, suggesting that at lower volumes of TPP, lesser -PO43− groups were available to cross-link with -NH3+; however, this circumstance overturned as the TPP volume increased. More -PO43− groups were present to interact with -NH3+ groups to form CNP, hence increasing the percentage of utilized -NH3+. A similar corresponding trend was also perceived previously by Masarudin et al. (2015) and this data correlated with particle size and dispersity of synthesized CNP as observed in Dynamic Light Scattering (DLS) analysis [The percentage of utilized -NHanalysis .43− to cross-link with -NH3+. However, the particle size and PDI value lessened as the TPP volume was increased as more -PO43− became available to perform internal cross-linking with -NH3+ within the CS particles, thus forming a more compact CNP and lower agglomeration rate. These results further supported the TNBS analysis.Based on Synthesis of nanoparticle sized between 50 nm to 100 nm, a range volume of 200 µL to 250 µL TPP for CNP-F1 A, 150 µLCollectively, formation of stable and monodispersed CNP within the size range of 50–100 nm, was only be attained from two CS: TPP volume ratios of 3:1 and 2.4:1 of CNP-F1. CNP-F1 at 3:1 CS: TPP volume ratio parameter yield an average CNP particle size distribution of 80.89 ± 5.16 nm and a PDI value of 0.26 ± 0.01, while at 2.4:1 CS: TPP volume ratio produced a CNP of 77.36 ± 13.99 nm and 0.22 ± 0.05 PDI value. A consistency in small-sized, stable, monodispersed CNP with a low PDI value was perceived from using 200 µL TPP and 600 µL CS in CNP-F1 and was In this study, CS concentration below 1.5 mg/mL and TPP below 1.0 mg/mL was used as better CS solubility and CNP stability was attained . Similar43−) and hydroxide ions (OH-) [3+ are deprotonated by anionic TPP, which leads to the formation of smaller and monodispersed CNP [43− and OH- [In aqueous conditions, TPP dissociates into phosphate ions (POns (OH-) . Studiesns (OH-) . Besidesrsed CNP ,61,62. C and OH- ,61,62. T and OH- .3+ of CS chains aid encapsulation of the compound within the core of CNP° via electrostatic interaction [The nutraceutical and antioxidant potential of CGA is overshadowed by poor compound stability and poor intracellular permeability. Administration of high dosage to encounter these deficits lead to many unexpected side effects ,35,64. Teraction . SuccessA prominent expansion in particle size was observed in CNP°-CGA samples as shown in Following the increase in CGA concentrations used for loading into CNP°; 2 µM, 10 µM, and 20 µM, CGA-EE% (percentage of CGA successfully loaded into nanoparticle) showed a declining trend from 74.43 ± 0.31%, 62.30 ± 0.05%, and 60.21 ± 0.03%, respectively, while CGA-L showed otherwise; 1.49 µM, 6.23 µM, and 12.04 µM, respectively  and Pavanveena et al. (2010), one factor that influences the amount of drug being entrapped within the nanoparticle is the drug to polymer ratio ,70. The Irregular, spherical-shaped and well dispersed CNP° and CNP°-CGA accompanied by particle size range of 70–80 nm and above 100 nm, respectively, were observed through Transmission Electron Microscopy (TEM) analysis in The Field Emission Scanning Electron Microscopy (FESEM) images of CNP° and CNP°-CGA in The particle size distribution result of FESEM slightly differ from the DLS analysis  as DLS m2 stretching vibration of CS at 3353.91 cm−1 was observed in CNP° at 3364.97 cm−1 and CNP°-CGA at 3398.61 cm−1 possibly due to presence of intermolecular hydrogen bonds during the formation of nanoparticles and encapsulated nanoparticles [−1.The functional groups attributed to FTIR spectrum of CS, TPP, CNP°, CGA, and CNP°-CGA were illustrated in articles ,76,77. W−1 and asymmetrical stretching vibration of P-O-P peak at 883.70 cm−1 were also apparent in CNP° at 1073.91 cm−1 and 948.77 cm−1, respectively, and in CNP°-CGA at 1014.92 cm−1 and 949.14 cm−1 respectively [−1) and N-H bending vibration in amide II (1587.00 cm−1) was identified in CNP° at peak 1630.94 cm−1 and peak 1527.82 cm−1, respectively, which was also perhaps due to TPP and CS cross-linking [Interestingly, two characteristic peaks of TPP; P=O stretching peak at 1126.89 cmectively ,77,78,79ectively ,77,80,81−1, C=O stretching vibration of carboxyl and ester group at 1687.62 cm−1, stretching vibration of C-O-C and C-O of carboxyl and ester group at 1279.45 cm−1 and 1185.17 cm−1, bending of C-H and COH at 1119.50 cm−1, and CH aromatic bending at 810.46 cm−1 of CGA spectrum were spotted in CNP°-CGA spectrum at 1428.52cm−1, 1700.27 cm−1, 1265.04 cm−1 and 1228.71 cm−1, 1163.43 cm−1, and 812.13 cm−1, respectively [The encapsulation of CGA in CNP° vector was discerned via the presence of five peaks attributed to CGA in the CNP°-CGA spectrum, which initially were absent in the CNP° spectrum. Aromatic ring C=C stretching vibration at 1439.88 cmectively ,87,88.Additionally, an increase in transmittance percentages of the N-H bending of amide II peak in CS, 52.24%, and P-O-P asymmetrical stretching vibration peak in TPP, 22.73% was observed upon formation of CNP°, 72.08% and 49.86%, respectively; may indicate the ionic interaction between TPP and CS. However, scarcely any changes in transmittance percentage of those two functional groups were perceived in CNP°-CGA , implying the energy transmittances were not affected by the electrostatic interactions between CGA and CS during the formation of CGA-nanoparticle hybrids. Hence, this supports the successful formations of CNP° and CNP°-CGA.The ability of CGA and CNP°-CGA to reduce DPPH into DPPH-H form was measured and compared to access their respective antioxidant strength. The common standard reference, ascorbic acid, was used. The concentration for all three samples, CGA, CNP°-CGA and ascorbic acid were limited to 12 µM (on the graph) as the CGA-EE% as 60.21 ± 0.03%, assuming approximately 60% of CGA was successfully encapsulated. As shown in Ascorbic acid reached its scavenging effect plateau point at 11 µM, with 70% of DPPH scavenged. Remarkably, similar scavenging plateau point was observed at CNP°-CGA; suggesting a maximum of 11 µM of CNP°-CGA was required to scavenge 0.1 mM DPPH—as can be observed from ortho position of its catechol; as claimed by Valgimigli et al. (2008) [CGA compound could actively partake in scavenging activity due to its two hydroxyl groups at . (2008) . However. (2008) ,94. This. (2008) ,96 and t50 values of pure CGA and CNP°-CGA, the minimum antioxidant concentration needed to scavenge 50% of DPPH radicals, are shown in 50 value of 6.5 µM similar to CGA, while CNP°-CGA displayed a slightly lower IC50 of 5 µM. We deduced that the encapsulated CGA has a better antioxidant strength as its scavenging activity was still conserved, and comparatively less CGA concentration was needed to scavenge 50% of DPPH than CGA to achieve similar scavenging activity.The ICfCNP°-CGA) in 786-O cell line was studied through fluorescent microscopic and flow cytometry analyses. FITC has the ability to conjugate with the primary amine group of CS through covalent bonding without dissociating and provides a sensitive study on cellular accumulation efficiency of fluorescently labeled nanoparticles under the influence of time  [fCNP°-CGA in 786-O cells was time-dependent, as the fluorescent intensity increased consequently with incubation time. Cellular uptake of fCNP°-CGA was detected as early as 30 min (fCNP°-CGA being taken up by 786-O cells. The fluorescent intensity in the cells thereafter increased in 6 h (Time-dependent in vitro uptake of FITC labeled CNP°-CGA (nd 48 h) ,97. Supps 30 min  post-tred in 6 h , 24 h , Jia et al. (2009), and Masarudin et al. (2015) [Conversely, smearing of the green fluorescent within the cells was observed at 24 h, and 48 h post-treatment, possibly due to the dissociation of FITC from CNP°-CGA . It also. (2015) ,52,110.fCNP°-CGA. The peak continued to shift and percentage increased as the incubation period was prolonged to 6 h (99.89%) . Hypnotizing that the uptake of fCNP°-CGA could prolong till cells reaches the saturation point without the instrument limit. The 48 h post-treatment of FITC only peak overlapped with the control peak (cells only) as shown in Complimenting fluorescent microscopic analysis, flow cytometry analysis was performed as shown in (99.89%) D, 24 h ((99.89%) E, and 48(99.89%) F; signifOne of the main deficits of CGA is its poor cellular permeability, halting this antioxidant-rich compound from exhibiting it chemopreventive mechanism. The hypothesis whereby nanoparticles could possibly aid this compound to improve its cellular permeability was proven through these two analyses, the microscopic viewing and flow cytometry analysis. These two studies are very crucial to demonstrate the facilitation of CNP° in internalizing CGA into cells. There is the possibility that as more CGA compounds are internalized within the cells, it could carry out its chemopreventive activity more efficiently without being expelled from the system as described by Olthof et al. 2001), Gonthier et al. (2003), and Farah et al. (2008) in their respective studies , Gonthie,29.Furthermore, multiple studies have supported the idea that the uptake of drug-nanoparticles hybrids were mainly influenced by particle size and the cellular uptake route. In this study, CNP°-CGA were synthesized with an average size of 134.44 ± 18.29 nm which falls within the favorable size range (50–200 nm) for an efficient cellular uptake ,68 most The MTT mediated cytotoxic assay was performed to study and compare the in vitro toxicity properties of the synthesized nanoparticles and drug-nanoparticles. Based on The naturally existing antioxidant-rich polyphenolic, CGA ,114 has Evidently, studies have reveal the ability of CGA to inhibit NF-κB transcription factor, repress DNA methylation, induce Nrf2 mediated antioxidant and detoxification enzymes (such as glutathione S-transferases (GST) and NAD(P)H: quinone oxidoreductase 1 (NQO1)) through antioxidant response element (ARE) to reduce oxidative stress cellular damages, and so on Reference ,117,118.Contrariwise, CGA is also known for its poor cellular permeability profile, loses its reliability as a potential nutraceutical compound ,28,29. COn the other hand, a significant shift in cytotoxicity effect on 786-O cell lines was observed following encapsulation of CGA in CNP° vector. The concentrations of CGA and CNP°-CGA used were again limited to 12 µM because of the maximum CGA-EE% of 60%. On the whole, the cell viability decreased in dose-dependent modus upon 24 h post-treatment of CNP°-CGA . Approxi50 of CGA and CNP°-CGA were approximately calculated; 68.80 µM and 16.20 µM, respectively. An approximately 4-fold more CGA concentration was needed to achieve similar cytotoxic effect as CNP°-CGA. Deducing the encapsulated CGA potentially reduced the need for high dosage administrations of CGA to the cell line.The shift in cytotoxic effect of CGA following encapsulation in nanoparticles was possibly due to positive surface charge of the vector contributed by the unconjugated protonated amine groups, and this cationic charge was hypothesized to be conserved even after encapsulating CGA. Additionally, this feature of the vector enables it to participate clathrin-mediated endocytosis route to permeate across the anionic cell membrane ,121,122.All materials were purchased and used without further purification, unless otherwise stated. Low molecular weight chitosan (CS) powder, sodium tripolyphosphate (TPP) powder, sodium dodecyl sulfate (SDS) powder, 2,4,6-trinitrobenzene sulfonic acid (TNBS) powder, L-ascorbic acid powder, 1,1-Diphenyl-2-picrylhydrazine 97%, (DPPH) powder, fluorescein 5(6)-isothiocyanate (FITC) powder, thiazolyl blue tetrazolium bromide (MTT) powder, phosphate buffered saline (10x) (PBS) solution, dimethyl sulfoxide (DMSO) solution, ethylenediaminetetraacetic acid (EDTA), and sodium carbonate powder, were purchased from Sigma-Aldrich . Hydrochloric acid solution, sodium hydroxide pellet, and glacial acetic acid solution, and chlorogenic acid (CGA) powder were purchased from Friendmann Schmidt Chemicals (Germany). Methanol solution, reagent grade AR was purchased from QReC . RPMI medium 1640 (1×) solution, Fetal Bovine Serum (FBS) solution, antibiotic-antimycotic (100×) solution, and 0.25% Trypsin-EDTA (1×) solution were purchased from Gibco, Life Technologies .2O), while TPP stock solution was prepared simply by dissolving 25 mg of TPP powder in sdH2O. The working solutions of CS and TPP were diluted to three different concentrations as shown Chitosan nanoparticles (CNP) were prepared based on modified ionic gelation method of Calvo et al. (1997) . One mg/3+) of CS and anionic phosphate groups (-PO43−) of TPP cross-linker. A slightly modified TNBS  assay protocol of Masarudin et al. (2015) was adapted to quantify the percentage of unconjugated primary amine group (-NH3+) in CNP formulations [3) at pH 8.5, 10% sodium dodecyl sulfate (SDS), 1 M hydrochloric acid (HCl), and 0.05% TNBS in 0.1 M NaCO3 at pH 8.5 were used for this assay. Serially diluted CS solution using NaCO3  were used as standards. CNP solutions were prepared based on the formulations in 3 at pH 8.5, 100 µL 10% SDS, and 75 µL of 0.1 M HCl solution was set as blank. The samples were analyzed at A335 using microplate reader . The percentage of free amine group was quantified using the following formula;CNP was formed via ionic gelation between protonated primary amine groups (-NHulations . Freshly2O at 60:40 (sdH2O: CNP) ratio before analyzing them using DLS. The best CNP formulation (denoted hereon as CNP°) was used for the subsequent analysis.The formation of nanoparticles, particle size distributions and PDI value of the synthesized CNP samples were measured via dynamic light scattering (DLS) analysis using a Malvern Zetasizer Nano ZS instrument . The prepared CNP samples were centrifuged at 13,000 rpm for 20 min and diluted in sdH2O. The CGA was then mixed with the best CNP formulation; CNP° to form CNP encapsulated CGA (CNP°-CGA). The particle size and PDI value of CNP°-CGA were analyzed via DLS method. While, to determine encapsulation efficiency (EE%), the CNP°-CGA was prepared and centrifuged at 13,000 rpm, for 30 min. The free, unloaded CGA were collected and the absorbance was measured at A325 using spectrophotometer (Thermo Scientific Genesys 840-208100 UV/Vis Spectrophotometer). The percentage of encapsulation efficiency  and CGA loading  were calculated using the following formulas;Chlorogenic acid (CGA) of 10 µM, 50 µM, and 100 µM initial concentration were prepared by dissolving the commercially purchased CGA powder in sdHThe best CNP°-CGA parameter was then subsequently used in the following analyses.To discern the formation hybrid nanoparticles, Field Emission Scanning Electron Microscopy (FESEM) and Transmission Electron Microscopy (TEM) were employed. The best parameter of CNP° and CNP°-CGA were prepared as described earlier. The samples were placed on a stub and dried in incubator at 37 °C for two to three days. The samples were then coated with gold and viewed under Ultra High Resolution FESEM . The CNP° and CNP°-CGA images were then processed using ImageJ software to assess the particle size distribution. The size of CNP° and CNP°-CGA particles were measured, and a histogram bar on diameter and normal distribution curve were plotted. While, for TEM analysis, both the samples were immobilized on copper grids and air dried before examining under TEM  at MIMOS Berhad.FITR analysis was performed to study the molecular components and chemical functional groups present in a sample, in this case, CS, TPP, CGA, CNP°, and CNP°-CGA. All samples were prepared fresh and were freeze dried before sending them for FTIR analysis at Faculty of Sciences, UPM. The obtained FTIR spectrum results were further analyzed.517nm using a microplate reader .The free radical scavenging activity of CGA, and CNP-CGA were evaluated using a modified DPPH radical scavenging assay protocol described previously by Blois, 1958 . CNP°, C2), and upon 80% to 100% confluency, the cells were used for subsequent cell treatment analysis or sub-cultured.Human renal adenocarcinoma cells, 786-O were obtained from ATCC (USA). The cell lines were grown and maintained in a complete growth media, CGM . The adherent cells were incubated at 37 °C, and 5% of carbon dioxide  in CGM and a 100 µL of each diluted samples were mixed into each well and the plate was left to incubate at 37 °C, 5% CO2 for 24 h. The untreated well was used as control, and well with media only was used as blank. After 24 h, the samples were discarded, and replaced with 170 µL of fresh CGM, and 30 µL MTT (5 mg/mL in 1× PBS). The plate was covered with aluminium foil and incubated for 4 h at 37 °C. Subsequently, 150 µL of the solution from each well was discarded prior to addition of 100 µL of DMSO to solubilize the formazan crystals. The absorbance was immediately read at A570nm using a microplate reader. The percentage of cell viability was calculated using the following formula;MTT assay was used to measure the cytotoxicity of the CGA, CNP°, and CNP°-CGA on 786-O cells. The 786-O cells were seeded on a 96 well plate at a volume of 100 µL, and a density of 10 × 10fCNP°-CGA was used in the following analyses.The primary amine groups of CS were covalently conjugated to 0.25 mg/mL of fluorescein isothiocyanate (FITC), which was dissolved in DMSO. The solution was incubated for 15 min. Successively, CGA, and TPP were added into the solution. Formed 5 cells/per well, and incubated for 24 h at 37 °C and 5% of CO2. The following day, the CGM was removed; the cells were rinsed with 1x Phosphate Buffered Saline (PBS), and fresh CGM were added into the wells. The cells were treated with FITC only for 48 h, and fCNP°-CGA in dark at different time points; for 30 min, 6 h, 24 h, and 48 h. The non-treated cells were used as control. The plate was covered with aluminium foil to avoid light exposure. After each time point, the samples were removed from the wells, rinsed with 1× PBS, and 2 mL of fresh CGM was added into each well. The cells were observed under fluorescent microscope, while for flow cytometry analysis, after each time point, the samples were discarded from the wells, rinsed with 1× PBS, and trypsinized. Upon trypsinization, the cells from each well were collected into individual tubes, centrifuged at 1000 rpm for 5 min. The pellets obtained were suspended in 1 mL of MASC buffer  before analyzing in a NovoCyte flow cytometry .Human renal adenocarcinoma cells, 786-O were seeded on 6 well plate at a density of 1 × 10n = 3) ± standard deviation (SD). The statistical significant of data was evaluated using Dunnett’s multiple comparison test, One Way Analysis of Variance (ANOVA), GraphPad Prism; at probability value less than 0.05 (P < 0.05), with degree of significant denoted with asterisk (*) symbol on.Triplicate independent reads were measured for all the analyses, and expressed as mean  at pH 5 and 200 µL TPP (0.7 mg/mL) at pH 2; with an average particle size of 80.89 ± 5.16 nm and a polydispersity index (PDI) of 0.26 ± 0.01.Formation of stable, monodispersed, chitosan nanoparticle was achieved through spontaneous ionic gelation between cationic CS and anionic TPP. Nanoparticles obtained were under 100 nm in size, and TPP volume was found to greatly influenced formation of CNP across different formulations; CNP-F1 (0.5 mg/mL CS and 0.7 mg/mL TPP), CNP-F2 (0.25 mg/mL CS and 0.35 mg/mL TPP), and CNP-F3 (0.1 mg/mL CS and 0.2 mg/mL TPP). At lower TPP volumes, inadequate -PO3+ of CS. The optimal parameter in synthesizing CGA loaded CNP° was attained at 200 µL of 100 µM CGA , 600 µL of 0.5 mg/mL CS, and 200 µL of 0.7 mg/mL TPP. CNP°-CGA formation using 100 µM CGA led to a prominent increase in CNP° particle size (an increase of 66.20%) with a conserved particle stability and monodispersity (PDI 0.29 ± 0.03) as compared to encapsulation of 10 µM (an increase of 13.72%) and 50 µM (an increase of 2.11%). The significant CGA-L, µM at this optimal parameter was recorded as the highest, 12.04 µM, despite the lowest CGA-EE%, 60.21 ± 0.03% compared to the other two concentrations.Encapsulation of the phytochemical CGA into CNP° was initiated through electrostatic interactions between CGA and protonated -NH50: 5 µM) was slightly better compared to CGA alone (IC50: 6.5 µM), and was able to scavenge the same amount of free radicals at lower CGA concentrations compared to CGA. Apart from that, both CNP° and CGA showed non-toxic properties towards human renal adenocarcinoma, 786-O cells, with percentage of cell viability of 81.37 ± 0.84% and above 100%, respectively. Inversely, CNP°-CGA prompted a better cytotoxic efficacy by reducing cell viability to 60.22% (at 12 µM) after 24 h post-treatment. In addition, the in vitro cellular  accumulation study disclosed that cellular internalization of fluorescently labeled CNP°-CGA begun as early as 30 min and proceeded up to 48 h post-treatment. Inferring, the cationic feature of CNP° shell secured and facilitated the uptake of CGA across the cell membrane.The DPPH scavenging efficacy of CNP°-CGA (ICThe synthesized CNP° vector is a good drug delivery system (DDS) candidate for CGA cargo by increasing its in vitro accumulation, thus, improving the bioavailability of CGA and enhancing its antioxidant and cytotoxic properties; hence, potentially lowers the administrative CGA dosages. Furthermore, positive surface charge of CNP° benefited as DDS by accommodating negatively charged drugs, by providing protective shell for the payload, and facilitating the permeability of encapsulated payload across the cellular membrane.Hitherto, researchers have been widely adapting CNP as a safe option for DDS, and CGA as a therapeutic compound in pharmaceutical industries. CNP° vector plays an importance role in delivering CGA across the membrane, which in turn influences the cellular uptake of CGA, increases its bioavailability, conserves its therapeutic efficacy, and reduces possible occurrence of side effects by reducing the administration dosage of CGA. Nevertheless, the molecular level mechanisms of CGA therapeutic efficacy and CNP as vector, stability of CNP°-CGA, release kinetics of CNP°-CGA, and antioxidant activity, in both in vitro and in vivo studies have yet to be explored further. These in-depth studies are crucial to the design of an efficient chitosan nanoparticle mediated delivery system that could potential protect and enhance the therapeutic efficacy of CGA as a treatment for chronic disease, specifically cancer.