T, was isolated during a study targeting the culture-dependent microbial diversity occurring in bentonite formations from southern Spain. Comparative 16S rRNA gene sequence analysis showed that BII-R7T represented a member of the genus Stenotrophomonas (class Gammaproteobacteria), and was related most closely to Stenotrophomonas rhizophila e-p10T (99.2 % sequence similarity), followed by Stenotrophomonas pavanii ICB 89T (98.5 %), Stenotrophomonas maltophilia IAM 12423T, Stenotrophomonas chelatiphaga LPM-5T and Stenotrophomonas tumulicola T5916-2-1bT . Pairwise sequence similarities to all other type strains of species of the genus Stenotrophomonas were below 98 %. Genome-based calculations  indicated clearly that the isolate represents a novel species within this genus. Different phenotypic analyses, such as the detection of a quinone system composed of the major compound ubiquinone Q-8 and a fatty acid profile with iso-C15 : 0 and anteiso-C15 : 0 as major components, supported this finding at the same time as contributing to a comprehensive characterization of BII-R7T. Based on this polyphasic approach comprising phenotypic and genotypic/molecular characterization, BII-R7T can be differentiated clearly from its phylogenetic neighbours, establishing a novel species for which the name Stenotrophomonas bentonitica sp. nov. is proposed with BII-R7T as the type strain (=LMG 29893T=CECT 9180T=DSM 103927T).A Gram-stain negative, rod-shaped, aerobic bacterial strain, BII-R7 T, affiliated to the genus Stenotrophomonas  on the basis of 16S rRNA gene sequence divergence , with a probability of equal to or above 70 % DDH of 0.1 %. All other comparisons resulted in lower dDDH values  followes et al.  and ANI istently . The dig species . BII-R7TH values . DNA G+Cll cases . In the Stenotrophomonas phylogenetically most closely related to BII-R7T  were compared with BII-R7T. The other distantly related remaining species of the genus Stenotrophomonas were not included in this comparative survey. Certain cultural, physiological, chemotaxonomic and biochemical key features of these strains of members of the genus Stenotrophomonas were analyzed, even if these analyses had been performed in previous studies, in order to guarantee a comprehensive comparative study.Based on DNA-based comparisons presented above, the four species of the genus 2O2, and oxidase activity by using a 1 % (w/v) solution of tetramethyl-p-phenylenediamine , moderate amounts of spermine [8.5 µmol (g dry weight)−1] and traces [<0.2 µmol (g dry weight)−1] of cadaverine, putrescine and 1,3-diaminopropane. This polyamine pattern was very similar to that of S. chelatiphaga DSM 21508T which has also been found to contain spermidine as the major polyamine [78.8 µmol (g dry weight)−1], moderate amounts of spermine [6.2 µmol (g dry weight)−1] and traces [<0.2 µmol (g dry weight)−1] of cadaverine, putrescine and 1,3-diaminopropane. On the other hand, the absence of significant amounts of cadaverine distinguish BII-R7T and S. chelatiphaga DSM 21508T from other species of the genus Stenotrophomonas. S. rhizophila DSM 14405T was found to have a polyamine pattern consisting of the major polyamines spermidine [87.1 µmol (g dry weight)−1] and cadaverine [17.5 µmol (g dry weight)−1] and moderate amounts of spermine [4.5 µmol (g dry weight)−1] and the polyamine pattern of S. pavanii DSM 25135T contained the major polyamines spermidine [58.3 µmol (g dry weight)−1] and cadaverine [16.3 µmol (g dry weight)−1], small amounts of spermine [2.3 µmol (g dry weight)−1] and traces [<0.2 µmol (g dry weight)−1] of putrescine. The polyamine pattern with high amounts of cadaverine resembles that of S. maltophilia which has been reported to show a pattern with almost equal amounts of cadaverine and spermidine  at 28 °C. Polyamines were extracted from biomasses harvested at the late exponential growth phase according to the protocol of Busse and Auling . HPLC eqet al.  and cond [et al. . The polermidine .et al. [T and the reference species showed the presence of the major lipid diphosphatidylglycerol, moderate amounts of phosphatidylglycerol and phosphatidylethanolamine and minor amounts of the unidentified aminophospholipid APL1. In the case of BII-R7T and S. chelatiphaga DSM 21508T their polar lipid profiles were almost identical, showing only some small quantitative differences in the lipids detected. Also, S. rhizophila DSM 14405T was highly similar but some minor lipids were not detected in this species . S. pavanii DSM 25135T was distinguishable from BII-R7Ton the basis of the presence of unidentified lipids including glycolipid GL1, aminolipid AL1, lipid L4 and phospholipid PL2. S. maltophilia DSM 50170T could be distinguished from BII-R7T by the presence of the unidentified lipids aminolipid AL2 and the two lipids L5 and L6 and absence of lipids L1, L2 and L3.Quinones and polar lipids were extracted from biomass harvested at the stationary growth phase applying the integrated protocol of Tindall  and Alteet al. . The HPLet al. . The quiT was consistent with the profiles described for species of the genus Stenotrophomonas as shown in 15 : 0 and anteiso-C15 : 0 and a variety of iso-branched hydroxylated fatty acids, typical of representatives of the genus Stenotrophomonas  were supported by a number of phenotypic differences, such as growth inhibition in the presence of 5 % NaCl, lack of cell motility and ability to reduce nitrates to nitrites.BII-R7features . Althougposition , motilitposition . In the a et al. . 16S rRNT represents a novel species of the genus Stenotrophomonas, for which the name Stenotrophomonas bentonitica sp. nov. is proposed.On this basis, BII-R7Stenotrophomonas bentonitica .2. Catalase, leucine arylamidase, protease, esterase, esterase lipase, acid and alkaline phosphatase, naphtol-AS-BI-phosphohydrolase and β-glucosidase activities are positive but oxidase activity is negative. No indole production occurs. The strain is able to utilize the following carbon sources: d-xylose, cellobiose, glutarate, melibiose, p-arbutin, N-acetyl-d-galactosamine, N-acetyl-d-glucosamine, d-glucose, maltose, d-mannose, acetate, propionate, fumarate, dl-lactate, malate, pyruvate, d-ribose, salicin and trehalose. The predominant fatty acids are iso-C15 : 0 and anteiso-C15 : 0. In addition a variety of iso-branched hydroxylated fatty acids are produced. The polyamine pattern contains the major polyamine spermidine, moderate amounts of spermine and traces of cadaverine, putrescine and 1,3-diaminopropane. The quinone system is composed of the major compound ubiquinone Q-8 and small amounts of Q-7. The polar lipid profile contains the major lipid diphosphatidylglycerol, moderate amounts of phosphatidylglycerol, and phosphatidylethanolamine and minor amounts of an unidentified aminophospholipid, a phospholipid and three lipids.Cells are Gram-stain negative, aerobic, non-motile, with no flagella and do not form endospores. When grown on LB agar at 28 °C for 3 days, colonies are light yellow, smooth, convex and circular. Under these growing conditions, cells are straight rods 0.45–0.65 µm in width and 0.92–1.55 µm in length and occur singly or in pairs. Growth takes place at 15–37 °C but not at 4 or 40 °C (optimum is 28 °C) at pH 5–10 but not at pH 4 or 11 (optimum at pH 7) and with 0–2.5 % NaCl but not with 5 % NaCl. The organism is able to reduce nitrate to nitrite but not to NStenotrophomonas bentonitica BII-R7T (=LMG 29893T=CECT 9180T=DSM 103927T), and was isolated from bentonite formations. The DNA G+C content of the type strain is 66.5 mol%.The type strain is


N-heterocyclocarbenic ligands. Our mechanistic study reveals the charge-neutral generation of polaron pairs  by electron transfer from the dopant to host excitons. Annihilation of the radical ion pair occurs by charge recombination, with such annihilation competing with bond scission. Device lifetime correlates linearly with the rate constant for the annihilation of the radical ion pair. Our findings demonstrate the importance of controlling exciton-induced electron transfer, and provide novel strategies to design materials for long-lifetime blue electrophosphorescence devices.Degradation of organic materials is responsible for the short operation lifetimes of organic light-emitting devices, but the mechanism by which such degradation is initiated has yet to be fully established. Here we report a new mechanism for degradation of emitting layers in blue-phosphorescent devices. We investigate binary mixtures of a wide bandgap host and a series of novel Ir(III) complex dopants having  The short lifetime of blue-phosphorescent organic light-emitting devices owing to material degradation impedes their practical potential. Here, Kim et al. study the molecular mechanism of the degradation that involves exciton-mediated electron transfer as a key step for the generation of radical ion pairs. Extensive research has enabled organic light-emitting devices (OLEDs) to outperform conventional displays in various commercial applications. One widely recognized remaining challenge, however, is to improve device lifetime. In particular, the short operation time of devices that emit blue light are significant impediments to exploiting the full potential of OLEDs.4. The mechanism by which organic materials degrade involves radical species as byproducts and key reaction intermediates10. Once formed, the open-shell radical species deteriorates device performance by trapping charge carriers or by quenching light emission through exciton–polaron annihilation or nonradiative recombination21. Previous studies established exciton-localized homolysis and imbalance in charge carrier concentrations to be responsible for the generation of the radical species25. Molecules containing weak covalent bonds undergo homolysis in the excited state, producing neutral radical species30. Charged radical species  are generated inevitably via charge carrier injection and transport during normal device operation. Efforts have thus been made to minimize the population of radical species within emitting layers by decreasing the density of excitons or by suppressing the buildup of charge36.Growing evidence has ascribed the short device lifetime to an accumulation of chemical defects resulting from irreversible degradation of organic materials during device operationDespite these advances, there has been a limited understanding of the fundamental reason for the significant instability of devices that emit blue light. Note that an intermolecular pathway for radical generation has been overlooked. It is possible that the exciton is reductively quenched by electron transfer from a nearby molecule with a shallow oxidation potential to form a radical ion pair , due to the positive driving force of the electron transfer Fig. . Such rakBeT) are determined through second-order kinetics analyses. Multilayer OLEDs are fabricated, and a linear correlation is found between kBeT and device lifetime. The study reveals the importance of controlling the electrochemical potentials of a host exciton and its dopant for achieving long device lifetimes.In the current research, we investigate a charge-neutral, exciton-induced generation of radical ion pairs between a wide bandgap host and blue-phosphorescent Ir(III) complex dopants. The radical ions and their annihilation processes in the host–dopant binary system are directly monitored for the first time, with employing a variety of chemical techniques. The rate constants for charge recombination by back electron transfer (N-heterocyclocarbenic (NHC) ligands (Ir1–Ir4) were newly synthesized and employed as blue-phosphorescent dopants. 3,3′-Biscarbazolyl-5-cyanobiphenyl (H) was used as a wide-bandgap host material37. Chemical structures of the Ir dopants and H are depicted in Fig. Eox) and −1.82 V (Ered) vs. SCE, respectively  as large as 3.10 eV was determined, which enabled calculation of the excited-state oxidation (E*ox) and reduction (E*red) potentials of H according to the relationships E*ox = Eox − ΔEg and E*red = Ered + ΔEg. In this way, E*ox and E*red of H were calculated to be −1.60 and 1.28 V vs. SCE, respectively. Comparison of the E*red value of H with the oxidation potentials of the Ir dopants revealed positive driving forces (−ΔGeT) in the range 0.16−0.47 eV for the reductive quenching of an H exciton through forward one-electron transfer from the dopant, where −ΔGeT was calculated from the equation −ΔGeT = e⋅[Eox(Ir) − E*red(H)]   can be estimated from the equation −ΔGBeT = e·[Ered(H) − Eox(Ir)]. The −ΔGBeT values were calculated to be as large as 2.63−2.94 eV  complexes having igands Ir–Ir4 wereon) Fig. . This th eV Fig. , suggestigands Ir–Ir4 were12 M−1 s−1. Although energy transfer to Ir3 cannot be excluded, the observation that sensitized phosphorescence of Ir3 due to energy transfer from H decreased with increasing equimolar concentrations of Ir3 and H suggested a concentration-dependent generation of non-emissive [H•−Ir•+]  with the addition of the Ir3 dopant  species of Ir•+. These peaks disappeared upon cessation of photoirradiation, which indicated that charge recombination occurred. Taken together, the results revealed that a radical ion species formed through rapid electron transfer.Photoinduced electron paramagnetic resonance (EPR) spectroscopy provided direct evidence for the formation of radical species. As shown in Fig. •−  during oxidative electrolysis at 1.30 V vs. SCE , which was determined from the spectroelectrochemical measurements. The kBeT values determined for the other Ir dopants were 4.5 × 1010 M−1 s−1 (for Ir1), 3.0 × 1010 M−1 s−1 (for Ir2), and 2.8 × 1011 M−1 s−1 (for Ir4). The kBeT values corresponded well to the theoretical curves obtained from the classical Marcus theory for adiabatic outer-sphere electron transfer, with the reorganization energies in the ranges 1.7–1.9 eV and 2.2–2.3 eV for the benzimidazole-based NHC Ir(III) complexes (Ir1 and Ir2) and the imidazopyrazine-based NHC Ir(III) complexes (Ir3 and Ir4), respectively . The UV–vis absorption spectrum of Ir4 showed a characteristic intense band at 390 nm due to an intraligand charge transfer (ILCT) transition localized within the NHC ligand. The absorbance of the ILCT transition band decreased during the photolysis -N,N′-diphenyl--4,4′-diamine), mCP benzene), DBFPO -dibenzofuran), and Liq (lithium quinolinate) served as a hole-injection layer, a hole-transporting layer, an electron-blocking layer, an electron-transporting and hole-blocking layer, and a buffer layer, respectively. The emitting layer consisted of a 30-nm-thick film of H that was molecularly doped with 10 or 20 wt % Ir dopant. The electroluminescence spectra  were indeed calculated to be greater than their triplet exciton energy (ΔET) values , allowing for the exciton recovery. Such exciton recovery, however, was inferred to be disallowed for Ir1 and Ir2, since their −ΔGBeT values  were calculated to be less than their ΔET values . A summary of obtained electroluminescence data for the H:Ir devices is shown in Supplementary Table Having established the degradation mechanism, we attempted to correlate the electron-transfer kinetics with device lifetime. Multilayer blue-phosphorescent OLEDs were fabricated using vacuum evaporation techniques with an ITO/HAT–CN/NPB/mCP/emitting layer/DBFPO/Liq/Al device configuration Fig. . HAT–CN tra Fig.  were idetra Fig. , excludiIr1 Fig. . While w−2. Figure LT70, which corresponds to the operation time when the luminance decreases to 70% of its initial value, approached 93.05 h for the 20 wt % Ir4 device, and decreased in the order Ir4, Ir3 (19.70 h), Ir2 (4.11 h), and Ir1 (1.88 h). The dissimilarity between this LT70 order and the order of the triplet state energy value of the Ir dopants was interpreted as excluding exciton-localized homolysis of the dopant from being a dominant degradation pathway. It is worth noting that the LT70 value was observed to increase in proportion with kBeT biphenyl) in place of H as a host material. It is predicted that radical ion pairs are more accumulated in the mCBP:Ir layers than the H:Ir layers, due to the more positive E*red  and similar Ered  values of mCBP. Indeed, devices of mCBP exhibited poor longevity consistently for all the Ir dopants, as quantitated from shorter LT70 values  facilitate more rapid formation of radical ion pairs, but much larger driving forces for back electron transfer (−ΔGBeT) impede charge recombination occurring in the Marcus-inverted region of electron transfer. The combined effect is a longer lifetime of radical ion pairs in an emitting layer of a blue-phosphorescent OLED than those of green-phosphorescent and red-phosphorescent OLEDs. It should be noted that accumulated levels of the radical ion species are minimal, because the forward electron transfer competes with energy transfer to a dopant, and, also, rapid charge recombination occurs. Overall, our mechanistic study revealed that exercising the electrochemical control to minimize the density and lifetime of radical ion pairs is crucial for realizing long operation lifetimes of OLEDs.We have investigated exciton-induced generation of radical ion pairs between a wide bandgap energy host and blue-phosphorescent Ir(III) complexes having NHC ligands. Spectroscopic techniques were employed to detect charge-neutral production of a pair of the radical cation of the dopant and the radical anion of the host. In particular, photoinduced EPR experiments and nanosecond laser flash photolysis provided direct evidence for the formation of the radical cation of the dopant molecule. Charge recombination kinetics within the radical ion pair was analyzed by employing the second-order kinetics model. Charge recombination was found to occur in the Marcus-inverted region of electron transfer. Analyses of the radical ion-mediated degradation products revealed the occurrence of oxidative cleavage of the dopant and reductive degradation of the host. Finally, a strong linear proportionality was found between the device lifetime and the rate constant for charge recombination. This finding is valuable, because it provides reasonable explanations for the poor stability of OLEDs that emit specifically blue light. Here, large driving forces for electron transfer  were recorded using a Bruker, Ultraflex III TOF/TOF 200 spectrometer. The host molecule (H) was prepared following the method reported previously1. Purity of the Ir dopants was examined by performing high performance liquid chromatography (HPLC) analyses. A Waters, Alliance e2695 was employed for the HPLC measurements ; eluent, acetonitrile:water = 70:30 ; sampling solvent, THF; oven temperature, 40 °C; flow rate, 1.0 mL min−1; APCI mode). LC-mass analyses were carried out to validate the HPLC results, employing a Shimadzu, LCMS–IT–TOF instrument. Elemental analyses were also performed for C, H, and N on Thermo Fisher Scientific, Flash1112 or Flash2000 instruments. H was prepared following the method reported previously37. Organic materials for device fabrication were purchased from commercial suppliers, and purified by sublimation at 10−5 torr prior to deposition. Films were deposited under high vacuum (<1.0 × 10−6 torr) onto a pre-cleaned glass substrate (1 cm × 1 cm). Poly(methyl methacrylate)  films doped with the Ir dopants (15 wt %) were dissolved in 1, 2-dichoroethane . The solution was sonicated for 30 min, and passed through a membrane filter (pore size = 8.0 μm). An aliquot of the polymer solution was placed onto a pre-cleaned glass substrate, and was spin-casted with employing an EPLEX, SPIN‐1200D spin coater. Spectrophotometric grade THF stored under an insert atmosphere was used for spectroscopic and electrochemical measurements.Chemicals were purchased from commercial suppliers, and used without further purification. d]imidazol-3-ium iodide , silver(I) oxide , and IrCl3·3H2O  was refluxed for 16 h under an Ar atmosphere. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel using dichloromethane as an eluent. Recrystallization in dichloromethane and n-hexane gave Ir1 as a pale yellow powder in a 57% yield (1.10 g). 1H NMR : δ 8.30 , 8.17 , 7.70 , 7.44 , 7.42–7.36 , 7.32 , 7.00 , 6.77 , 3.39 ; 13C{1H} NMR : δ 190.09, 150.41, 148.71, 143.03, 137.57, 136.97, 134.86, 133.15, 129.28, 127.16, 126.74, 123.77, 123.45, 122.65, 111.89, 111.50, 110.57, 34.25; MALDI–TOF/MS (m/z): [M]+ calcd. for C60H45IrN6, 1042.33; found, 1042.49; analysis : C , H , N .A DMF solution containing 1--3-yl)-3-methyl-1H-benzo[d]imidazol-3-ium iodide  in place of 1--3-yl)-3-methyl-1H-benzo[d]imidazol-3-ium iodide. A pale yellow powder . 1H NMR : δ 8.21 , 7.96 , 7.39–7.29 , 7.32−7.26 , 7.20 , 7.12 , 7.00 , 3.39 ; 13C{1H} NMR : δ 189.84, 149.79, 149.24, 142.99, 137.14, 136.95, 135.98, 133.13, 128.80, 127.23, 126.40, 123.34, 122.51, 120.62, 112.76, 111.80, 110.41, 34.18, 30.28; MALDI–TOF/MS (m/z): [M]+ calcd. for C60H45IrN6, 1042.33; found, 1042.56; analysis : C , H , N .The method identical to that for Ir1 was employed, except the use of 1--4-yl)-3-methyl-1H-benzopyrazin-3-ium iodide , silver(I) oxide , and chloroiridium(I) dimer  was refluxed for 20 h under an Ar atmosphere. After cooling to room temperature, the solvent was removed in vacuo. The crude product was purified by column chromatography on silica gel, using dichloromethane/n-hexane  as an eluent. Recrystallization in dichloromethane/n-hexane gave Ir3 as a yellow crystal . 1H NMR : δ 8.80 , 7.13 , 6.87–6.81 , 6.77 , 6.61 , 6.48 , 2.72 , 2.43 ; 13C{1H} NMR : δ 191.87, 147.64, 147.11, 145.76, 144.67, 141.13, 138.24, 136.82, 136.63, 128.84, 128.47, 128.06, 125.85, 122.24, 115.31, 22.46, 22.17; MALDI–TOF/MS (m/z): [M]+ calcd. for C57H45IrN12, 1090.35; found, 1090.58; analysis : C , H , N .An tert-butyl)phenyl)-5,6-dimethyl-1H-imidazopyrazin-3-ium iodide  in place of 5,6-dimethyl-1,3-diphenyl-1H-imidazopyrazin-3-ium iodide. A yellow crystal . 1H NMR : δ 8.76 , 6.83 , 6.75 , 6.63 , 6.31 , 2.66 , 2.43 , 1.35 , 1.18 ; 13C{1H} NMR : δ 192.96, 152.28, 147.51, 144.98, 144.07, 141.25, 138.01, 136.74, 135.38, 127.72, 125.41, 125.12, 122.71, 112.46, 35.05, 34.80, 31.98, 31.49, 22.49, 22.14; MALDI–TOF/MS (m/z): [M]+ calcd. for C81H93IrN12, 1426.73; found, 1427.01; analysis : C , H , N .The method identical to that for Ir3 was employed, except the use of 1,3-bis, 340 nm (Ir2), 396 nm (Ir3), 400 nm (Ir4), and 340 nm (H). The PLQYs were determined employing an absolute PLQY measurement system .τobs values of the Ir dopants and H host. Photoluminescence decay traces were acquired based on time-correlated single-photon-counting (TCSPC) techniques using a FluoTime 200 instrument . A 377 nm diode laser  was used as the excitation source. The burst and normal modes embedded in the Time Harp 260 P module  were employed for acquiring the signals from the Ir dopants and H host, respectively. The photoluminescence signals were obtained at 467 nm (Ir1 and Ir2), 460 nm (Ir3), 474 nm (Ir4), and 400 nm (H), through an automated motorized monochromator. Photoluminescence decay profiles were analyzed  using a single exponential decay model.Ar-saturated 50 μM solutions in THF were used for the determination of the 3 couple was used as a pseudo reference electrode. Measurements were carried out in Ar-saturated THF (2.0 mL) using 0.10 M tetra-n-butylammonium hexafluorophosphate (TBAPF6) as the supporting electrolyte at scan rates of 0.10 V s−1 (cyclic voltammetry), 4.0 mV s−1 , and 25 mV s−1 . The concentration of the Ir dopants and H host was 2.0 mM. A ferrocenium/ferrocene couple was employed as the external reference. The potentials were reported as values against SCE, by adding 0.257 V. In the case of negative scans, validity of the observed peaks was examined by comparing voltammograms for a blank solution.Cyclic, differential pulse, and second harmonic alternating current voltammetry experiments were carried out using a CH Instruments, CHI630 B instrument equipped with a three-electrode cell assembly. A Pt wire and a Pt microdisc were used as the counter and the working electrodes, respectively. An Ag/AgNO3 for Ir1, 0.65 V vs. Ag/AgNO3 for Ir2, 0.95 V vs. Ag/AgNO3 for Ir3, and 0.80 V vs. Ag/AgNO3 for Ir4), using the amperometric I–t curve method. A blank spectrum was taken for a 0.10 M TBAPF6 solution (THF) in a spectroelectrochemical cell (path length = 0.5 mm) equipped with a Pt mesh working electrode, a Pt wire counter electrode, and an Ag/AnNO3 pseudo reference electrode. 500 μL of a 2.0 mM Ir dopant solution was delivered into the spectroelectrochemical cell for the measurement.UV–vis–NIR absorption spectra of the radical species were obtained on an Agilent, Cary 5000 spectrophotometer with applying the anodic potentials  × 1/(1 − 10Abs−) where Iobs, Abs, and Abs0 are the observed fluorescence intensity, the absorbance of H at 300 nm in the presence of Ir, and the absorbance of H at 300 nm in the absence of Ir, respectively. The I0/I values were fit to the Stern–Volmer equation I0/I = (1 + Ka·[Ir]) × (1 + kq·τ0·[Ir]). In this equation, Ka, kq, τ0, and [Ir] are the association constant, the quenching constant, the fluorescence lifetime in the absence of Ir (3.8 ns), and the molar concentration of Ir, respectively.Fluorescence quenching experiments were performed for a THF solution containing 100 μM H with added Ir (0–60  μM). Photoexcitation wavelength at 300 nm produced fluorescence emission of H, exclusively. Therefore, a monotonic decrease in the H emission was observed upon increasing the concentration of the Ir dopant. The decrease was quantitated as 2(5p)6] electrons and the (5d)6 valence electrons. Frequency calculations were subsequently performed to assess the stability of the convergence. Time-dependent density functional theory (TD–DFT) calculations were carried out for the optimized geometries using the same functional and basis sets. Geometry optimization and single-point calculations were performed using the Gaussian 09 program39. GaussSum was employed for simulation of the predicted electronic absorption spectra40.Geometry optimization was performed using Becke’s three-parameter B3LYP exchange-correlation functional modified with the Coulomb-attenuated method (CAM–B3LYP), the double-ξ quality LANL2DZ basis set for the Ir atom, and the 6−311 + G basis set for all the other atoms. A pseudo potential (LANL2DZ) was applied to replace the inner core electrons of the Ir atom, leaving the outer core , in the −ΔGBeT range 0–3.0 eV. In this equation, Z, kB, and T are the collisional frequency taken as 1.0 × 1012 M−1 s−1, the Boltzmann constant, and absolute temperature (298 K), respectively.An Ar-saturated THF solution in a quartz cell (path length = 1.0 cm) was excited by a Nd:YAG laser  at a wavelength of 355 nm with 20 mJ pulseAn Ar-saturated THF solution (3.0 mL) containing 3.0 mM H and 100 μM Ir was photoirradiated under a broad-band light from a Xenon lamp  for 10 min. A color change from yellow to orange was observed during the photolysis, which was monitored through steady-state UV–vis absorption spectroscopy. Photolysis was also performed for vacuum-evaporated films of H molecularly dispersed with 10 or 15 wt % Ir (glass substrate). The films were photoilluminated under 325 nm  or the broad-band light from the Xenon lamp, during which the changes in the photoluminescence (325 nm irradiated films) or UV–vis absorption (broad-band illuminated films) spectra were monitored. PMMA films molecularly doped with Ir dopants (15 wt %) were prepared and served as controls.3CN , and passed through a membrane filter (pore size = 8.0 μm) prior to injection. A 5 μL was injected and allowed to pass through the column at room temperature, using an eluent gradiently increased fractions of CH3CN in H2O. Chromatographic detection was performed with employing a UV detector (λobs = 254 nm). Electrospray ionization mass analyses were subsequently performed at a positive ion detection mode (voltage = 70 V) in the range 200–1500 a.m.u. The photoirradiated films of H and Ir were dissolved in CH3CN (HPLC grade) for the HPLC analyses.HPLC experiments were performed on an Agilent, 6120 DW LC/MSD instrument equipped with a Poroshell, EC-C18 column. The photolyzed solutions were diluted in HPLC grade CH−6 torr. A 1-nm-thick Liq layer and a 100-nm-thick Al layer were deposited as a cathode through thermal evaporation. The deposition rates of the organic and metal layers were 0.1 and 0.5 nm s−1, respectively. Deposition of Liq was carried out at a rate 0.01 nm s−1. The active device area of 4 mm2 was defined by the area of an overlap between the ITO and Al electrodes. Current, voltage, and luminance of the devices were measured with a system consisting of a Keithley, 2400 Source-Mete,r and a PR-650 spectroradiometer. Operational lifetime measurements of the devices were taken in a constant current mode. LT70 values were determined from the decay traces of % luminance plotted as a function of operation time. Operation time at which the % luminance decreased to 70% corresponded to LT70.The organic layers used were deposited consecutively on pre-cleaned ITO glass substrates by employing a thermal evaporation system at a pressure <1.0 × 10The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information(PDF 2094 kb)Peer Review File(PDF 1094 kb)


Marinomonas sp. BSi20414), was successfully purified to homogeneity from Marinomonas sp. BSi20414 isolated from Arctic sea ice by ammonium sulfate precipitation and anion exchange chromatography, resulting in an 8.12-fold increase in specific activity and 9.9% recovery in total activity. MaBGA displayed its maximum activity at pH 6.0 and 60 °C, and maintained at least 90% of its initial activity over the pH range of 5.0–8.0 after incubating for 1 h. It also exhibited considerable thermal stability, which retained 76% of its initial activity after incubating at 50 °C for 6 h. In contrast to other β-galactosidases, MaBGA displayed strict substrate specificity, not only for the glycosyl group, but also for the linkage type. To better understand the structure–function relationship, the encoding gene of MaBGA was obtained and subject to bioinformatics analysis. Multiple alignments and phylogenetic analysis revealed that MaBGA belonged to the glycoside hydrolase family 42 and had closer genetic relationships with thermophilic β-galactosidases of extremophiles. With the aid of homology modeling and molecular docking, we proposed a reasonable explanation for the linkage selectivity of MaBGA from a structural perspective. On account of the robust stability and 1,3-linkage selectivity, MaBGA would be a promising candidate in the biosynthesis of galacto-oligosaccharide with β1–3 linkage.A novel β-1,3-galactosidase, designated as MaBGA (β-galactosidase from  The enzyme β-galactosidases EC 3.2.1.23, BGA), which are widely distributed in various organisms, including animals, plants, bacteria, archaea, yeasts and fungi, are capable of catalyzing the hydrolysis of molecules containing the β-glycosidic bond, to release their terminal non-reducing galactose molecules. In some cases, β-galactosidases can catalyze the reverse reaction of the hydrolysis, transglycosylation, when receptors of galactosyl are monosaccharides, disaccharides or oligosaccharides, instead of water molecules  and mK are the concentration and Michaelis constants of ONPG, respectively.For steady-state kinetic analysis, the activity of MaBGA was measured by using various concentrations of ONPG from 0.1 mM to 19 mM. The kinetic constants of the enzyme were determined by using a nonlinear fitting of the Michaelis–Menten equation: −1.To determine the activity of MaBGA toward different linkage types, Galβ1–3GlcNAc, Galβ1–4GlcNAc and Galβ1–6GlcNAc were used as substrates, respectively. The reaction products were filtered by nitrocellulose membrane with pore size of 0.22 μm, in advance of being subject to detection by HPLC equipped with an Aminex HPX-87P column and differential detector. The column temperature and flow rate were set as 85 °C and 0.5 mL·minmabga was amplified by using degenerate primer pairs F1/R1, F1/R2, F1/R3, F2/R1, F2/R2, F2/R3, F3/R1, F3/R2, F3/R3, A208/B1, A208/C1, A208/C2, A76/B1, A76/C1, A76/C2, A195/B1, A195/C1 and A195/C2  strategy [mabga gene.The 5′ and 3′ flanking regions of the known partial sequence were obtained by using DNA Walking strategy . The neshttp://www.ncbi.nlm.nih.gov/BLAST). Alignment of multiple protein sequences was conducted using the Clustal X 2.0 program [Homologous search in GenBank was performed using the BLAST server ( program  and rend program . A phylo program  in MEGA6 program , with a The three-dimensional model of MaBGA was constructed by using MODELLER , RobettaThe refined model was used for docking with Galβ1–3GlcNAc, Galβ1–4GlcNAc and Galβ1–6GlcNAc, respectively, using Autodock 4.2  with defMarinomonas sp. BSi20414, was first purified to homogeneity and characterized extensively. MaBGA displayed robust stability against heat and strict substrate specificity toward both the glycosyl group and the linkage type. Although further experiments are required to decipher its substrate recognition mechanism, our study provided an attractive alternative for biosynthesis of galacto-oligosaccharide with β1–3 linkage and laid the groundwork for the protein engineering to modify the linkage preference of β-galactosidase.In this work, a thermostable β-1,3-galactosidase MaBGA derived from


CD4+ and CD8+ T lymphocytes reside in the epithelium and lamina propria of the penile regions and have mostly a resting memory phenotype. All penile regions contain CD56dim NK cells surface expressing the natural cytotoxicity receptor NKp44 and the antibody-dependent cell cytotoxicity receptor CD16. These cells are also able to spontaneously secrete pro- and anti-inflammatory cytokines, such as IL-17 and IL-22. Finally, CCR10 is the main homing receptor detected in these penile cells although, together with CCR3, CCR6, and CCR9, their expression level differs between penile compartments. Unlike antigen-presenting cells which type differ between penile regions as we reported earlier, urethral, fossa, and glans content in immune B, T, and NK cells is comparable. However, median values per each analysis suggest that the glans, containing higher number and more activated NK cells together with higher number of terminally differentiate effector CD8+ T cells, is a superior effector site than the urethra and the fossa. Thus, the human penis is an immunologically active tissue containing the cellular machinery required to induce and produce a specific and effective response against mucosal pathogens. It can therefore be considered as a classic mucosal effector site, a feature that must be taken into account for the elaboration of efficient strategies, including vaccines, against sexually transmitted infections.The human penis is a main portal of entry for numerous pathogens, and vaccines able to control resulting infections locally are highly desirable. However, in contrast to the gastrointestinal or vaginal mucosa, the penile immune system and mechanisms inducing a penile immune response remain elusive. In this descriptive study, using multiparametric flow cytometry and immunohistochemistry, we characterized mucosal immune cells such as B, T, and natural killer (NK) cells from the urethra, fossa, and glans of human adult penile tissues. We show that memory B lymphocytes and CD138 Chlamydia trachomatis  are a main health issue worldwide , and thechomatis  or Neissorrhoeae . Furtherorrhoeae –10.To reduce or prevent these STIs, vaccine strategies targeting the penis are crucially needed. Accordingly, initial HIV-1 vaccine studies were able to induce HIV-1 specific mucosal antibodies, although non-neutralizing, in the male genital mucosa . FurtherThe human penis consists of four different regions: (i) the foreskin, a stratified keratinized epithelium, with a highly keratinized outer face and a less keratinized inner one facing the glans , (ii) th+ T cells help, and the cellular response driven by cytotoxic T cells.Innate and adaptive immune responses contribute both to protection at mucosal surfaces . The mucPenile mucosal immune cells and their interactions with STI have been little studied due to the difficulty in obtaining human tissues, whereas the foreskin immunity is better understood particularly in the context of HIV-1 infection. Hence, we showed that HIV-1 targets first LCs during sexual transmission of HIV-1 in non-circumcised men , providiThe study was performed according to local ethical regulations, following approval by the local ethical committee . Written informed consent was provided by all study participants.Whole penile tissues were obtained from 39 individuals undergoing elective gender reassignment surgery at the Saint Louis Hospital in Paris, France . Hormonal treatment was stopped 2 months before surgery, and all patients lacked history of STIs during the 6 months before the surgery. Tissues were transported to the laboratory immediately after surgery in phosphate-buffered saline (PBS) supplemented with 20 μg/ml gentamicin (Gibco) and used within the next 2 h.g.Urethra, fossa, and glans were first mechanically separated, each region cut into 8 mm × 8 mm pieces, and the underlying fat and muscle removed. To prevent denaturation or removal of cell surface molecules by dispase/trypsin enzymes that are routinely used to separate the epidermal and dermal compartments, cells were directly extracted from the tissues by collagenase type IV digestion  in the presence of DNAse I  for 2 h at 37°C . After e6 cells/well) and labeled with indicated combinations of antibodies  or matched isotype control antibodies  for 30 min at 4°C. Cells were washed twice in PBS-2% FBS and fixed with Cytofix/Cytoperm solution (BD Biosciences) for 15 min at room temperature.Cells were resuspended in PBS-2% FBS, transferred to a 96 round-bottom well plate  and analyzed with Kaluza Software (Beckman-Coulter). As shown on Figure Penile cells were plated in 500 µl culture medium (RPMI medium containing 10% FBS) for 5 h at 37°C in the presence of Brefeldin A . Cells were washed twice in PBS-2% FBS and labeled with anti-CD4, anti-CD8 and anti-CD56 antibodies or matched isotype control antibodies for 30 min at 4°C. Cells were washed twice in PBS-2% FBS and fixed with Cytofix/Cytoperm solution (BD Biosciences) for 15 min at room temperature. Cells were washed with PermWash buffer (BD Biosciences) and stained for intracellular IL-2, IFN-γ, TNF-α, IL-4, IL-5, IL-17, and IL-22 for 30 min at 4°C using appropriate antibodies  or corresponding isotype control antibodies , diluted in PermWash buffer.Data were acquired with an LSRII cytometer  and analyzed following the gating strategy shown in Figure Urethra, fossa, and glans tissue pieces were fixed with PBS-4% paraformaldehyde, embedded in paraffin and serially sectioned at 4 µm. Immunohistochemistry was performed as we previously described , 10 usinUrethra, fossa, and glans tissue pieces (5 mm × 3 mm × 1 mm) were lysed in 500 μl of PBS-0.2% SDS containing a protease inhibitor cocktail , disrupted using a Qiagen TissueRuptor. Resulting cell lysates were stored at −80°C. Cytokines, namely, MCP-1/CCL2, MIP-3α/CCL20, RANTES/CCL5, TRAIL/TNFSF10, MCP-4/CCL13, IL-4, IL-13, CTACK/CCL27, and Eotaxin/CCL11, were quantified using the Luminex technology  on a Bio-Plex 200  according to the manufacturer’s recommendations. Furthermore, CCL25 and CCL28 were quantified using the Quantikine immunoassay  according to the manufacturer’s recommendations.U-test. p-Values < 0.05 were considered significant. *p < 0.05, **p < 0.01, and ***p < 0.001.Statistical analysis was performed with Prism6 software (GraphPad). For flow cytometry analyses, tissues of the various penile regions did not always originate from the same individual. The results are expressed as median, followed by the 95% confidence interval (95% CI). For comparing the immune cell populations between the different penile compartments, although such statistics have to be taken cautiously due to the limited number of tissue samples, the non-parametric Kruskal–Wallis test was used. Pairwise comparisons were performed by the non-parametric Mann–Whitney B cells are an important population of adaptive immunity driving the humoral response by producing antibodies after antigenic stimulation. Characterization of their phenotypes is a fundamental prerequisite for the elaboration of efficient vaccine strategies against STIs.−CD19+ B cells represented 0.5–3.0% of CD45 cells, with median [95% CI range]: 3.2% [2.8–3.45] in the urethra, 2.3% [1.7–2.8] in the fossa, and 1.3% [0.7–2.0] in the glans.Therefore, the presence of B lymphocytes, defined by the expression of the pan leukocyte marker CD45 and CD19, but lack of CD3, was determined in each penile compartment by multiparametric flow cytometry Figures A,B. CD3−+CD27− naïve (BN), CD21+CD27+ resting memory (BRM), CD21−CD27+ activated memory (BAM), and CD21−CD27− tissue-like memory (BTLM) B cells  and fossa (64.5% [55.5–73.5]), it was higher in the glans (74.4% [70.9–77.8]). In addition, while BAM and BRM were both present in urethra  and fossa , BAM were significantly less abundant (19.3% [15.4–23.2] p = 0.0236 relative to the urethra), and BRM were almost absent  in the glans. Finally, BN were present at similar proportions in urethra (10.5% [8.6–12.4]), fossa (9.6% [7–12.2]), and glans (7.2% [5.8–8.5]).The major B cell subset was BTLM > BAM) predominate over resting memory/naive B cells in the penis.Altogether, memory B cells  cross-linking (+ B cells was higher in the urethra (20.3% [16.8–23.9]) compared with the fossa (10.6% [8.2–12.9]) and glans (5.7% [3.1–8.2]) ; (2.8% [1.8–3.7]); and (2.5% [1.3–3.7]) for urethra, fossa and glans, respectively] , fossa (4.2% [3.3–5.0]), and glans (1.8% [0.9–2.6])  and syndecan-1 together with their lamina propria localization are indicative of the presence of antibody-secreting PCs.+ and CD8+ T cells are key players in the adaptive immune response. To identify their presence and profile in penile tissues, the frequency of cells co-expressing CD3/CD4 and CD3/CD8 was determined amongst CD45+ population . CD8+ T cells were equally present in all penile regions   and anti-CD8 (brown staining) antibodies. Hence, CD8+ T cells co-express CD3 and CD8 (blue/brown staining), whereas CD4+ T cells expressed CD3 but not CD8 (blue staining only) , CD45RA−CCR7+ central memory (TCM), CD45RA−CCR7− effector memory (TEM), and CD45RA+CCR7− effector (TE) T cells. CD4+ T cells . TE constituted an important glans CD8+ subset (53.5% [49.8–57.1] of CD8+ T cells), whereas in the urethra and fossa, CD8+ TE dropped significantly .Next, T cell maturation was assessed using CD45RA and CCR7 to distinguish four T cell subsets , namely,, 95.1% 9.5–96.7 iIM) were next quantified based on CD27 expression in the CCR7 negative memory T cells population . Penile CD8+ T cells, as CD4+ T cells, had also a resting phenotype .Last, T cell activation, defined by co-expression of HLA-DR and CD38, was assessed Figure A. In the2.8% 1.2–.4   EM cells, CD8+ T cell subsets include mainly EM cells but also effector cells, especially in the glans. Furthermore, the higher expression of CD103 by CD8+ relative to CD4+ T cells translates into a higher epithelial CD8+ T cells distribution than that of CD4+ T cells, although both subsets are equally present in the lamina propria.Overall, CD4T helper (Th) cells represent one of the most important cell type in adaptive immunity, as they are required for almost all adaptive immune responses. Once activated, Th cells secrete different types of cytokines that in turn stimulate other immune cells such as B cells, which provide help to secrete antibodies or cytotoxic T cells for killing infected target cells, thereby amplifying the immune response against pathogens . Flow cy+ cells    and slightly more abundant in the glans (3.3% [2.2–4.3]) , the fossa (1.6% [1.4–1.8]), and the glans (1.7% [1.6–1.8])  , most likely because CD56 expression was too low for visualization by immunohistochemistry.In line with this CD56The main activating receptors of penile NK cells, namely, the members of the natural cytotoxicity receptors (NCR) family NKp30, NKp44, and NKp46 , were exIn addition to T cells, innate immune cells including NK cells also secrete the inflammatory cytokines IL-17 and IL-22 . We thus+-activated NK cells residing in the lamina propria that are equipped to trigger ADCC via the CD16 receptor, and are able to secrete spontaneously pro-inflammatory cytokines such as IL-17 and IL-22.Altogether, all penile tissue regions are equally populated by NKp44+CD8+ T cells were more numerous than CCR5+CD4+ T cells in the urethra (69.2% [58.5–79.8] vs 60.9% [49.4–72.4]), fossa (70.4% [58.2–82.5] vs 42.5% [28.9–55.9]), and glans (69.8% [59.4–80.1] vs 66.7% [58.8–74.6]). Furthermore, both CD4+ and CD8+ T cells expressed higher level of CXCR4 in the urethra  compared with CD4+ and CD8+ T cells from fossa  and glans .We first evaluated CCR5 and CXCR4 expression on T cells Figures A,B, two +, and CD8+ T cells in all penile regions , MIP-3α/CCL20 (one ligand of CCR6), CCL25 (the ligand of CCR9), CTACK/CCL27 and CCL28 (the ligands of CCR10), RANTES/CCL5 (one ligand of CCR5), TRAIL/TNFS10 (a member of the tumor necrosis factor family of ligands capable of initiating apoptosis through engagement of cell death receptors), and the inflammatory cytokines IL-4 and IL-13 using the luminex and quantikine technologies , whereas RANTES, IL-13, and CCL28 concentrations were 100 times higher . MCP-1, TRAIL, and CCL25 concentrations were intermediate (14.0–44.1 pg/ml mg). In fossa and glans  which could explain their epithelial localization throughout the penile regions. Accordingly, this homing receptor retains T cells in genital epithelia but also in other mucosa, such as the gut, by interacting with E-cadherin expressed by epithelial cells  Île-de-France XI, approval no. 11016]. All subjects gave written informed consent in accordance with the Declaration of Helsinki.AS, FR, YG, and MB conceived and designed the experiments; AS, FR, MD, SH, and DD performed the experiments; AS, FR, and MB analyzed data and wrote the article; MR and SC enrolled patients and peformed the surgery.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.


Plasmodium vivax are also treated with 14 days primaquine  (PQ). The aim of this study was to assess the efficacy of the national policy.First-line schizontocidal treatment for uncomplicated malaria in the Republic of the Sudan is artesunate  plus Sulphadoxine/pyrimethamine (25/1.25 mg/kg) (AS/SP). Patients with Plasmodium falciparum and/or P. vivax malaria were treated with AS/SP. Patients with P. falciparum were randomized to no primaquine (Pf-noPQ) or a single 0.25 mg/kg dose of PQ (Pf-PQ1). Patients with P. vivax received 14 days unsupervised 3.5 mg/kg PQ (Pv-PQ14) on day 2 or at the end of follow up (Pv-noPQ). Primary endpoint was the risk of recurrent parasitaemia at day 42. G6PD activity was measured by spectrophotometry and the Accessbio Biosensor™.Patients above 1 year, with microscopy-confirmed, P. falciparum (74.8%), 77 (24.9%) with P. vivax and 1 (0.3%) patient with mixed infection were enrolled. The PCR corrected cumulative risk of recurrent parasitaemia on day 42 was 3.8% (95% CI 1.2–11.2%) in the Pf-noPQ arm compared to 0.9% (95% CI 0.1–6.0%) in the Pf-PQ1 arm; . The corresponding risks of recurrence were 13.4% (95% CI 5.2–31.9%) in the Pv-noPQ arm and 5.3% (95% CI 1.3–19.4%) in the Pv-PQ14 arm . Two (0.9%) patients had G6PD enzyme activity below 10%, 19 (8.9%) patients below 60% of the adjusted male median. Correlation between spectrophotometry and Biosensor™ was low .231 patients with P. falciparum and P. vivax. The addition of PQ reduced the risk of recurrent P. falciparum and P. vivax by day 42, although this did not reach statistical significance. The version of the Biosensor™ assessed is not suitable for routine use.AS/SP remains effective for the treatment of Trial registrationhttps://clinicaltrials.gov/ct2/show/NCT02592408The online version of this article (10.1186/s12936-018-2266-9) contains supplementary material, which is available to authorized users. There has been a decline in the incidence of malaria in many sub-Saharan countries in the last decade –3, whichPlasmodium vivax , the risk being greater in patients treated with Pf-noPQ (3.4% [95% CI 1.1–10.3%]) compared to 0.9% (95% CI 0.1–6.0%) in those treated with Pf-PQ1; Hazard Ratio HR = 0.26 [95% CI 0.03–2.47], p = 0.203.In the P. falciparum had gametocytes detected on blood film examination during follow up.By day 42 the risk of parasite recurrence was 2.5% [95% CI 2.06–6.01%]; 4.8% (95% CI 1.8–12.4%) in the Pf-noPQ and 0.9% (95% CI 0.1–6.0%) in the Pf-PQ14 ; Fig. P. vivax arms, a total of 6 patients had recurrent parasitaemia at days 14 (n = 1), 21 (n = 1), 28 (n = 1), 35 (n = 3). In patients not receiving 14 days primaquine the risk of recurrent P. vivax at day 28 was 9.9% [95% CI 3.3–27.8%] compared to 0% in those treated with AS/SP plus 14 days primaquine; p = 0.046. At day 42 the respective risks had risen to 13.4% [95% CI 5.2–31.9%] in the Pv-noPQ arm and 5.3% [95% CI 1.3–19.4%] in the Pv-PQ14 arm; HR 0.36, [95% CI 0.1–2.0], p = 0.212  patients with P. vivax  of patients receiving a sub-optimal dose of any of the study drugs were excluded, leaving 127 (54.9%) of patients with 09 of patNo patient received sub-optimal AS Table . For patP. falciparum and 11.0 g/dl in patients with P. vivax , compared to patients infected with P. vivax whose Hb did not change  over the same period; p = 0.015 .At baseline the median Hb concentration was 11.7 g/dl in patients with on Table . On day P. falciparum the Hb concentration at day 7 had risen 6.0% (IQR − 0.8 to 14.8) from baseline following treatment with Pf-noPQ compared to 13.2% (IQR 0.0 to 23.9) after treatment with Pf-PQ1; p = 0.007. In patients with P. vivax, there was no change in Hb (IQR 0.0 to 10.0) between day 0 and day 7 following treatment with Pv-noPQ compared to 9.1% (IQR 0.0 to 15.4) in vivax patients treated with primaquine (Pv-PQ14); p = 0.106. None of the patients included required a blood transfusion.The median Hb levels increased in all treatment arms between days 0 and 7 Fig. , Table 4A total of 551 adverse events (AE) were reported, of which 497 (90.2%) were categorized as mild and 51 (9.8%) as moderate. Three (0.5%) mild events were recorded as possibly related to AS/SP. The most frequent AEs were gastrointestinal symptoms including poor appetite (40.3%), nausea (25.6%) and abdominal pain (20.7%)  patients had G6PD activity between 30 and 60%  patients. The adjusted male median by spectrophotometry was 9.7 U/gHb, compared to 7.9 U/gHb derived by the Biosensor™ (p < 0.001). By spectrophotometry 2 (0.9%) patients with 60% Fig. .Fig. 4G6s = 0.330 (p < 0.001), with a mean difference of 2.1 U/gHb and the 95% limit of agreement (LoA) ranged from − 5.1 U/gHb to 9.2 U/gHb  with activities between 10 and 30% activity, and 27 patients with G6PD activity between 30 and 60% , although these benefits did not reach statistical significance. Results on gametocyte carriage are limited by the small denominator applied for microscopy (200WBC).This efficacy study demonstrates that when the target dose of AS/SP was achieved the risk of recurrent parasitaemia was low for patients with either 25 mg/kg . While tP. falciparum [P. falciparum by day 28 generally exceeds 95%, except for one site in Gedaref, in the East of the country, where the efficacy fell from 91% in 2012 to 87% in 2015 [In 2006, a study in the east of the country reported a day 28 PCR corrected efficacy of 93.5% for AS/SP for lciparum . Other c in 2015 .pfdhfr and pfdhps genes and a single case with a triple mutation in the pfdhfr gene, all associated with antifolate resistance [One study from Kassala, Eastern Sudan reported a PCR corrected efficacy of 93.7% by day 28 , while asistance .P. falciparum [In Somalia, the efficacy of AS/SP has fallen to 88% and artemether–lumefantrine has replaced AS/SP as the first-line treatment for lciparum . In MarcP. falciparum and P. vivax AS/SP has been used as a universal schizontocidal treatment for both species particularly in locations where diagnostic facilities are unable to distinguish reliably between species. To date 3 published trials have assessed the efficacy of AS/SP against P. vivax. Tjitra et al. found AS/SP to have almost 90% efficacy against P. vivax in Papua, Indonesia [P. vivax by day 42 after AS/SP rose to 25% in Afghanistan [P. vivax recurrence at day 42 was 13.4% in patients treated with AS/SP alone, less than half that expected in our a priori power calculation. The risk of recurrence fell to 5.3% in those treated with AS/SP plus 14 days primaquine although this did not reach statistical significance (p = 0.212); Table In areas co-endemic for ndonesia . Howeverhanistan , and 67%hanistan . In the P. vivax can be achieved with a 14-day regimen of primaquine, although the long course of treatment is associated with poor adherence and compromised effectiveness [WHO guidelines recommend that the radical cure of tiveness , 40–44. tiveness , and indP. falciparum infection there was no further fall in Hb observed between day 2 and day 7, with or without a single dose of primaquine. These observations are reassuring and concur with previous reports from Tanzania [P. vivax infection had G6PD activity below 30%, although 2/22 (9.1%) had intermediate activity between 30 and 60%, reassuringly none of the patients had a significant fall in Hb after day 2.PQ is known to cause haemolysis in G6PD deficient individuals with the nadir usually observed 2–7 days after initial exposure . The greTanzania  supportiTanzania . None ofHowes et al. had earlier documented a country wide G6PD deficiency prevalence of 15.3% with higher frequencies towards the West of the country and lower frequencies in the East . These pThe study also evaluated a novel point of care Biosenor™  which was compared to the reference method of spectrophotometry. The results highlight that the current Biosensor™ underdiagnosed severe G6PD deficiency, a finding consistent with a recent similar study conducted in Bangladesh . Out of P. falciparum and a 14-day regimen in patients with P. vivax were well tolerated, and associated with a modest reduction in the number of recurrences by day 42, however reduction was not significant for P. falciparum. The Biosensor™ achieved suitable operational characteristics, but test performance is insufficient for routine diagnosis. A second generation with integrated haemoglobin measurement will be introduced to the market and may provide a more suitable solution.AS/SP appears to retain adequate efficacy in uncomplicated malaria in New Halfa and Gezira Slanj. Both a single dose of primaquine in patients with Additional file 1. Scatter plot body weight vs. total dose SP received. red line = target dose recommended by the WHO.Additional file 2. Database.


H1/TH2-type cytokines, serum levels of testosterone and estradiol were evaluated in F1 pups at four and eight weeks of age. The study showed that male pups were more sensitive to the effects of PFOS than female pups. At eight weeks of age, an imbalance in TH1/TH2-type cytokines with excess TH2 cytokines (IL-4) was found only in male pups. As for hormone levels, PFOS treatment in utero significantly decreased serum testosterone levels and increased estradiol levels only in male pups, and a significant interaction between sex and PFOS was observed for serum testosterone at both four weeks of age (pinteraction = 0.0049) and eight weeks of age (pinteraction = 0.0227) and for estradiol alternation at four weeks of age (pinteraction = 0.0351). In conclusion, testosterone-mediated endocrine function may be partially involved in the TH1/TH2 imbalance induced by PFOS, and these deficits are detectable among both young and adult mice and may affect males more than females.Little information exists about the evaluation of potential developmental immunotoxicity induced by perfluorooctane sulfonate (PFOS), a synthetic persistent and increasingly ubiquitous environmental contaminant. To assess potential sex-specific impacts of PFOS on immunological health in the offspring, using male and female C57BL/6 mice, pups were evaluated for developmental immunotoxic effects after maternal oral exposure to PFOS  during Gestational Days 1–17. Spontaneous T In female offspring at four weeks of age, splenic cellularity for those in the 5.0 mg/kg/day dam treatment group was significantly decreased compared against the values seen in control counter-parts [(0.74 ± 0.05) vs. (0.93 ± 0.07) × 108)]. At eight weeks of age, thymic and splenic cellularity was not changed relative to values in control offspring in either sex, save for thymic cellularity in male pups in the 5.0-mg/kg/day dam group [(1.07 ± 0.06) vs. (1.26 ± 0.09) × 108)].Treatment of dams during Gestation Days (GD) 1–17 with 0.1, 1.0 or 5.0 mg PFOS/kg/day resulted in a downtrend in splenic and thymic cellularity among male offspring at four weeks of age . For mal+CD8− population among all splenocytes was significantly reduced by 26% vs. control pup levels in four weeks of age male offspring of dams exposed to 5.0 mg/kg/day [(1.64 ± 0.09) vs. (2.21 ± 0.18) × 107)], and total levels of CD4−CD8+ cells was decreased by 20% [(8.49 ± 0.36) vs. (10.62 ± 0.61) × 106)]. Levels of double positive or double negative cells were not affected in male pups in any treatment group. In comparison, there were no significant effects on CD4−CD8+ or CD4+CD8− cell levels among any of the four weeks of age female offspring. In these hosts, only levels of double negative cells were significantly affected in the 5.0 mg/kg/day dam pups. Interestingly, in four weeks of age offspring of either sex in this highest dam-dose group, the absolute number of splenic B220+ cells was significantly altered from the values in corresponding controls.CD4/CD8 marker analysis on thymic and splenic lymphocytes from offspring was done to assess functional cell type population changes and stages of lymphocyte maturation in pups exposed in utero to PFOS. The FACS analyses of splenic T-lymphocytes  showed t+CD8− cells was still significantly reduced by 23% in the male offspring from dams exposed to 5.0 mg/kg/day [(1.37 ± 0.10) × 107 vs. (1.77 ± 0.09) × 107)], but the above-noted effects on CD4−CD8+ and B220+ cells was no longer evident. Among female pups from similarly-treated dams, only changes in B220+ cell levels were still evident  × 107 vs. control value of (4.41 ± 0.15) × 107).By adulthood (eight weeks of age), the total number of CD4−CD8− and a 23% decrease in CD4+CD8− cell levels compared to values in corresponding control pups. For the double negative populations, levels changed to (5.77 ± 0.50) × 107 from (7.44 ± 0.42) × 107; this effect was no longer apparent in eight weeks of age hosts. For the CD4+CD8− thymocytes, in the four weeks of age hosts, the values were significantly reduced to (1.37 ± 0.06) × 107 from (1.78 ± 0.09) × 107; and in the eight weeks of age mice, they were reduced to (1.53 ± 0.10) × 107 from (1.87 ± 0.08) × 107.With regard to thymic lymphocytic sub-populations , the onlMitogen-induced proliferative responses (B and T cell) among isolated splenocytes from offspring of dams exposed to PFOS (or vehicle) were measured to determine the effects on lymphocyte proliferation in these in utero-exposed pups. As shown in pfortrend = 0.003). At eight weeks of age, NK cell activity was decreased in male and female mice in both the 1.0 and 5.0 mg PFOS/kg/day groups  vs. (42.54 ± 2.17)) A. Althouy groups B. Specifpfortrend < 0.001). Activity was only significantly suppressed in cells from female pups from dams treated with 5 mg PFOS/kg/day (24% decrease from control). There were no significant effects on PFC response observed among cells from offspring of any dam treatment group when they attained eight weeks of age (The PFC responses (SRBC-specific IgM production by B-lymphocytes) of splenic cells obtained from four and eight weeks of age mice exposed in utero to PFOS were evaluated. As seen in s of age B.As shown in H1 and TH2-type cytokines assessed here was not markedly affected in cells obtained from the female mice in any dam treatment group. As for spontaneous production of IL-10 and IFNγ, although no statistically-significant associations were found with PFOS exposure in utero for either sex, a significant alteration trend was still observed (pfortrend < 0.05). For example, the p-value of the trend for IFNγ in female pups at four weeks of age was 0.047.At eight weeks of age, only cells that were obtained from male pups in the 5.0 mg PFOS/kg/day dam exposure group had a level of IL-4 production that was still significantly increased  vs. 15.32 (±1.88) pg/mL). In contrast, spontaneous production of all of the Tpfortrend = 0.229 for testosterone and pfortrend = 0.914 for estradiol, respectively; at eight weeks of age: pfortrend = 0.997 for testosterone and pfortrend = 0.599 for estradiol, respectively).The effects of PFOS on hormone levels in the serum of male and female pups are shown in p-value of the interaction term = 0.0049) and eight weeks of age (pinteraction = 0.0227). Furthermore, a positive effect of modification by sex was found for estradiol alternation at four weeks of age (pinteraction = 0.0351), but not at eight weeks of age (pinteraction = 0.1747). However, as for other parameters, such as for NK cell function, PFC levels, spontaneous production of IFNγ, IL4 and IL2, the effect modification was not significant at both four and eight weeks of age.In order to evaluate whether the effects were indeed stronger in males vs. females, we conducted tests of the interaction between sex and PFOS exposure for the functional parameters. The results showed that there was a significant interaction between sex and PFOS concentrations for serum testosterone alteration at both four weeks of age .6C3F1 pups flowing oral maternal exposure to PFOS on Gestational Days 1–17; their results showed that SRBC-specific IgM production was decreased in male pups after exposure to 5.0 mg/kg/day during gestation, but not altered in female pups  × 100).Offspring mice were euthanized at 4 and 8 weeks of age via COg ), and then, erythrocytes present were lysed with cold 0.17% (w/v) ammonium chloride (NH4Cl). Remaining cells were washed twice with medium, and then, each organ population of cells was re-suspended in RPMI 1640 for counting and assessment of viability . The cells were then diluted with complete RPMI 1640 or, depending on the experiment, aliquots were removed, centrifuged and re-suspended in PBS at concentrations outlined in the protocols below.The spleen and thymus from each offspring were passed through sterile fine-wire mesh with 10 mL complete RPMI 1640 bearing 5% heat-inactivated FBS, 25 mM HEPES, 0.12% gentamycin and 2 mM glutamine. Cell suspensions were centrifuged at 350× 2 and then re-constituted in 0.5 mL methanol. To remove any suspended materials and insoluble particles, the sample was then passed through a nylon filter . Using an Agilent1100 liquid chromatography/MSD SL mass spectrometry system , each extracted solution was analysed as previously described  × 100%, where E = the release of LDH  from target cells incubated in the presence of lymphocytes, S = the spontaneous release of LDH from target cells incubated in absence of lymphocytes and M = the maximum release of LDH determined by lysing the target cells with 1% NP-40.To determine NK cell activity among the splenocytes isolated from each offspring host, a lactate dehydrogenase (LDH) release assay was performed . For the6 cell/mL in complete RPMI 1640. Aliquots (50 µL) of the cells were then placed in Eppendorf tubes containing RPMI 1640 and SRBC (at 5%). An aliquot (50 µL) of lypophilized GPC re-constituted in GPC restoring solution and then diluted further (1:2) in GPC restoring solution was then added to the tube, and all the materials were loaded into Cunningham chamber slides. After incubation at 37 °C for 1 h, the slides were sealed and plaques on the slide and counted using a light microscope. Three slides per mouse were assessed, and the results were reported as PFC/106 splenocytes.A Cunningham modification of the Jerne plaque assay was used to determine the number of plaque-forming cells (PFC) among isolated splenocytes ,25,31. F5 cells) of cells were placed into wells of 96-well plates and then incubated for 48 h at 37 °C in a humidified 5% CO2-air chamber before samples of the culture supernatants were collected. Levels of interferon-γ (IFNγ), interleukin-2 (IL-2), IL-4 and IL-10 that were spontaneously produced/released by the splenocytes were then measured using ELISA kits, following the manufacturer protocols for each kit . The final OD value for each well was assessed at 450 nm using an Emax Microplate Reader . Concentrations of IL-2, IL-4, IL-10 or IFNγ in each sample were obtained by extrapolation from a standard curve prepared in parallel during each assay. The levels of sensitivity of the kits were 5 pg IL-2/mL, 5 pg IL-4/mL, 3 pg IL-10/mL and 1 pg IFNγ/mL.Aliquots  and homogeneity , and changes were made if needed. Transformations are outlined in the figure legends. A one-way analysis of variance (ANOVA) was used to determine differences among doses for each end-point using SAS software  in which pooled variance was used to determine the standard error. When significant differences were detected by the F-test (p < 0.05), a Dunnett’s t-test was used to compare treatment groups against the control group. Linear regression was used for the trend across dose. Analyses were sex-stratified, but we also investigated differences by sex, including the interaction of this variable with the PFOS exposure (sex × treatment) in the joint models by using a two-way ANOVA.All experiments were repeated twice  with


Blank formulation (RGD-MCMN) had z-average diameter of 162 ± 6 nm, polydispersity index of 0.11 ± 0.04, zeta potential of +13.2 ± 1.9 mV and (6.2 ± 1.1) × 1011 particles mL−1, while RGD-MCMN (Dox100) and RGD-MCMN (Dox500) showed respectively 146 ± 20 and 215 ± 25 nm, 0.10 ± 0.01 and 0.09 ± 0.03, +13.8 ± 2.3 and +16.4 ± 1.5 mV and (6.9 ± 0.6) × 1011 and (6.1 ± 1.0) × 1011 particles mL−1. RGD complexation was 7.73 × 104 molecules per nanocapsule and Dox loading were 1.51 × 104 and 7.64 × 104 molecules per nanocapsule, respectively. RGD-functionalized nanocapsules had an improved uptake capacity by U87MG cells. Pareto chart showed that the cell viability was mainly affected by the Dox concentration and the period of treatment in both MCF-7 and U87MG. The influence of RGD-functionalization on cell viability was a determinant factor exclusively to U87MG.Doxorubicin (Dox) clinical use is limited by dose-related cardiomyopathy, becoming more prevalent with increasing cumulative doses. Previously, we developed Dox-loaded lipid-core nanocapsules (Dox-LNC) and, in this study, we hypothesized that self-assembling and interfacial reactions could be used to obtain arginylglycylaspartic acid (RGD)-surface-functionalized-Dox-LNC, which could target tumoral cells overexpressing αvβ3 integrin. Human breast adenocarcinoma cell line (MCF-7) and human glioblastoma astrocytoma (U87MG) expressing different levels of αvβ3 integrin were studied. RGD-functionalized Dox-LNC were prepared with Dox at 100 and 500 mg·mL Cancer is a major public health problem worldwide. In the past decades, several nanoparticle-based drug delivery systems have obtained a great attention as promising approaches for tumor treatments ,2,3,4. TDoxorubicin (Dox), an anthracycline antibiotic and anticancer drug, is used as a cytostatic agent in cancer chemotherapy . UnfortuTo demonstrate efficacy, most of the anti-cancer drugs have to reach and internalize the cancer cells in a correct and appropriate concentration for an effective activity level . Moreovevβ3 integrin in angiogenesis and tumor cell proliferation is well documented . Hirsjärvi and co-workers [Angiogenesis, the development of new blood vessels, plays a critical role in controlling tumor growth and metastasis. Several cell surface-proteins, over-expressed by tumor endothelial cells, contribute to cell invasion and angiogenesis. Among them, the role of αcumented ,15. The  cancers ,17,18. T cancers ,19,20,21 cancers ,23,24. U-workers  developevβ3 integrin in different levels.In our research group, we developed a kind of polymeric nanocapsules named lipid-core nanocapsules (LNC), which structure is an organogel as core (sorbitan monostearate and capric-caprylic triglyceride), a polymer wall (composed of a hydrophobic polyester) and a hydrophilic corona of polysorbate 80 micelles . The lip11 particles mL−1, respectively. Formulation was prepared with an RGD concentration of 843.64 μmol L−1. After ultrafiltration-centrifugation, we determined the yield of RGD complexation on the surface of the nanocapsules as 94.3 ± 5.9%. In this way, the interfacial reaction yielded 7.73 × 104 RGD molecules per nanocapsule.The lipid-core nanocapsules were prepared by self-assembling methodology injecting an organic phase into an aqueous phase following evaporation to remove acetone and concentrate the formulation. The nanocapsules were, then, coated with chitosan. The chitosan-coated nanocapsule surface was reacted with zinc acetate and RGD. RGD-MCMN was obtained as macroscopically homogenous white-opalescent liquid. Photon correlation spectroscopy (PCS) analysis showed hydrodynamic mean diameter (z-average) of 162 ± 6 nm, polydispersity index (PDI) of 0.11 ± 0.04 and zeta potential of +13.2 ± 1.9 mV. By NTA, the hydrodynamic mean diameter and particle number density (PND) were 192 ± 10 nm and (6.2 ± 1.1) × 10In order to establish the influence of the RGD functionalization on the cell viability study, we prepared a formulation functionalized with phenylalanine (Phe), named here Phe-MCMN, as previously proposed as a control formulation in biological evaluations . The new−1. Therefore, the Dox-loaded nanoformulations functionalized with RGD were obtained as macroscopically homogeneous red-orange opalescent liquids. PCS and NTA showed nanoscopic populations, with mean diameters lower than 215 nm  and 92.61 ± 3.00% for RGD-MCMN (Dox500).Drug contents of 92.49 ± 7.17 and 457.55 ± 5.41 μg·mLf and RGD-MCMNf but in higher intensity for the latter in both cell cultures suggesting a higher RGD-MCMNf cellular uptake  affect the cell viability of MCF-7 and U87MG cells after 24 h of treatment. To MCF-7 culture, no significant difference was observed in cell viability after treatments with increasing concentrations of Phe-MCMN and RGD-MCMN compared to the control group . While, −4 and 2.06 × 10−4 μmol of nanocapsules per liter of well incubating them for 24 and 72 h. Dox-loaded nanocapsules were compared to Dox solution and the cell viability of the treated groups were calculated in relation to the control group (without treatment), which represents 100% of cell viability. During MTT assay, treated groups did not show significant alterations in the proliferation of the cell line when compared with the untreated group (control). After 24 and 72 h of treatment, all Dox treatments led to a significant decrease in cell viability in comparison with their respective control groups (p ≤ 0.05).To determine the cytotoxicity of Dox-loaded nanoformulations, we carried out the MTT assay applying the formulations at concentrations of 1.03 × 10−4 or 2.06 × 10−4 μmol of nanocapsules per liter of well. In the treatments with Doxorubicin concentration of 1.7 and 3.4 μmol·L−1, we used RGD-MCMN (Dox100), Phe-MCMN (Dox100) and Dox100, while for Doxorubicin concentration of 8.5 and 17 μmol·L−1 we used RGD-MCMN (Dox500), Phe-MCMN (Dox500) and Dox500. Taking into account the encapsulation efficiencies, the number Dox molecules per nanocapsule is about 1.51 × 104 or 7.64 × 104, respectively, for the formulations prepared using 100 and 500 μg of Dox per milliliter.MCF-7 cells were treated with drug concentration from 1.7 to 17 μmol of Dox per liter of well using two different concentrations of nanocapsules: 1.03 × 10p > 0.05) compared to the control (100% of viability). Furthermore, no statistical difference was determined comparing the formulations containing the highest concentration of Dox (p > 0.05). Nevertheless, regarding treatments with 1.7 and 3.4 μmol of Dox per liter of well using RGD-MCMN (Dox), a greater decrease in the viability of MCF-7 cells was observed compared to Phe-MCMN (Dox) (p < 0.05). MTT assay carried out with U87MG cells, after 24 h of treatment with similar Dox concentrations using the same formulations showed a cell viability from 51.45 ± 0.96% to 35.24 ± 0.51% for RGD-MCMN (Dox), from 54.43 ± 3.26% to 48.48 ± 0.94% for Phe-MCMN (Dox) and from 86.87 ± 5.24% to 61.10 ± 1.70% for Dox. The treatments using 1.7 and 3.4 μmol of Dox per liter of well did not show significant difference (p > 0.05) compared to the control (100% of viability). No statistical difference was determined comparing RGD-MCMN (Dox) and Phe-MCMN (Dox) using 1.7 and 3.4 μmol of Dox per liter of well (p > 0.05). In addition, nanocapsule formulations showed higher cytotoxicity than Dox in all concentrations of treatment (p < 0.05) and RGD-MCMN (Dox) showed the highest decrease of viability among the formulations.After 24 h of incubation, MTT assay  showed ap < 0.05) compared to the control. Only RGD-MCMN (Dox) demonstrated significant difference (p < 0.05) compared to other treatments in similar concentration. After 72 h, MTT assay carried out with U87MG cells, showed cell viability from 26.77 ± 3.21% to 3.21 ± 4.00% for RGD-MCMN (Dox), from 33.62 ± 4.17% to 20.93 ± 5.00% for Phe-MCMN (Dox) and from 65.89 ± 6.56% to 27.26 ± 2.16% for Dox. All treatments presented significant difference (p < 0.05) compared to the control. Dox presented significant difference (p < 0.05) compared to the treatments using 1.7 and 3.4 μmol of Dox per liter of well. RGD-MCMN (Dox) showed the highest decrease in cell viability compared to the other treatments using 17 μmol of Dox per liter of well (p < 0.05).After 72 h of incubation, MTT assay  in MCF-7To analyze the results considering all parameters (factors) varying among the groups of treatments, a Pareto chart was constructed . The facνβ3 integrin, which can specifically recognize the peptide RGD [In spite of much ongoing research about cancer, the treatment of tumors still proves difficult. Therefore, various nanoparticles based drug delivery systems have been studied ,27,29. Ttide RGD ,36,37,38tide RGD  describeνβ3) than similar concentrations of Phe-MCMN. In parallel, none of those formulations was cytotoxic to MCF-7 cells in similar experimental conditions.We developed the RGD-surface-functionalized metal-complex multi-wall nanocapsules by adapting the methodology we previously described  to obtai5 αvβ3 receptors per cell [Integrins are heterodimeric membrane glycoproteins composed of non-covalently associated α and β subunits. The αvβ3 integrin is highly expressed in U87MG cells, which express approximately 10per cell . Howeverper cell  the quanper cell  could noper cell .The supramolecular structure of the multi-wall nanocapsules is based on secondary bonds among the lipids and the lipids and PCL, as well as on the electrostatic interactions between the ammonium groups of chitosan and the negatively charge groups present in Lipoid S75 (lecithin). The addition of zinc acetate solution leads to the chitosan-metal complex formation. This complex is highly reactive ,42 and, EE%) was higher than 90% for both drug concentrations (100 or 500 μg·mL−1), demonstrating the high capacity of Dox loading on these nanocapsules. The high EE% in the present study is attributed primarily to the neutral drug form interacting with the lipid-core of the nanocapsules and also to probable electrostatic interactions between amine groups of Dox molecules and the oxygenated groups of Lipoid S75 (lecithin). Similarly, as previously described, Dox can be associated with the nanoparticles by electrostatic interactions [co-β-amino ester) and Dox-polymeric nanoparticles functionalized with RGD had higher cytotoxicity than nanoparticles prepared without RGD in U87MG cells [To the Dox-loaded nanocapsules, the encapsulation efficiency  showed promising results regarding the cytotoxicity in MCF-7 and U87MG cells. Those findings are likely explained by the aromatic residue of Phe as lateral chain, which can cause an increase in rupture capacity of the endosomal membrane as previously reported by Plank . FurtherPareto chart allowed confirming the importance of surface functionalization with RGD to decrease U87MG cell viability. In addition to this effect, Dox concentration and period of treatment were also significant to decrease cell viability for both tumor cells. Our hypothesis was validated. The significant effect of functionalization to inhibit the growth of U87MG cells is a consequence of the RGD targeting effect on these cells, which have specific integrin to bind RGD ,24,48,50f and Phe-MCMNf by both tumor cells. Therefore, the cellular uptake study showed that the increase in the nanocapsule concentration enhanced the fluorescence detected indicating a correlation to the cellular uptake. Besides, the similarity of Phe-MCMN and RGD-MCMN to MCF-7 cells viability, as well as the highest cellular uptake of RGD-MCMNf by U87MG cells corroborate to the Pareto chart analysis, which demonstrated the importance of the RGD functionalization to decrease the viability of U87MG cells. Wang and co-workers [vβ3 as well as RGD-nanoparticles and showed higher cellular uptake to B16F10 and MD-MB231 cells, with greater expression of αvβ3 integrin on the membrane.The fluorescence from nanocapsules was observed to U87MG and MCF-7 cells, showing the uptake of RGD-MCMN-workers  carried 2+, caused no significant changes in the cell death (apoptosis + necrosis) in both the RAW 264.7 macrophages and HUVEC cell lines [We previously investigated the toxicity of drug-unloaded LNC formulation prepared with PCL using in vivo models after oral and intraperitoneal administrations, respectively, to Caenorhabditis elegans  and Wistll lines . Even th® 60 (sorbitan monostearate), Poly(ε-caprolactone) , chitosan low molar weight , zinc acetate , QuantiPro™ BCA Assay Kit, 3--2,5-diphenyltetrazolium bromide (MMT) and arginylglycylaspartic acid (RGD) were obtained from Sigma-Aldrich . Caprylic/capric triglyceride and Tween® 80 (polysorbate 80) were delivered by Delaware . Lipoid® S75 (soybean lecithin) was obtained from Lipoid . Solvents used were of analytical or pharmaceutical grades. For cell culture studies, the human breast cancer cell line (MCF-7) and the human glioblastoma cell line (U87 MG) were obtained from the American Type Culture Collection . Dulbecco’s modified Eagle’s medium (DMEM low) and fetal bovine serum (FBS) were purchased from Invitrogen .Doxorubicin hydrochloride (98–102%), Span® water at 1 mg·mL−1. The orange-red solution was transferred to a separating funnel, added of trimethylamine (turning to violet) and extracted with chloroform (orange). The process was repeated until the aqueous phase was colorless. The organic phases were combined, dried under anhydrous sodium sulfate, filtered and evaporated under reduced pressure . Batches of 0.001 or 0.005 g were produced. The Dox (base) was dissolved in acetone and the solution was used to produce the formulations as described below.Dox (base) was obtained as previously reported . Briefly® 60 (0.040 g) and caprylic/capric triglyceride (0.120 g) were dissolved in acetone (25 mL) containing Dox (base) at 40 °C. Then, a solution of Lipoid S75® (0.090 g) in ethanol (7 mL) was added into the organic phase. This organic solution was injected into an aqueous phase (53 mL) containing Tween® 80 (0.080 g) under magnetic stirring at 40 °C. After 10 min, the organic solvents were evaporated and the formulation concentrated under reduced pressure at 40 °C using a rotary evaporator . A formulation containing no drug was also prepared as described above using acetone without Dox.Poly(ε-caprolactone) (0.100 g), Span−1 chitosan solution was prepared by dispersing chitosan in 1% acetic acid aqueous solution (MilliQ® water). The solution was filtered  and dropwise added (1 mL) into lecithin-polysorbate 80-coated lipid-core nanocapsule aqueous dispersion (9 mL) under magnetic stirring (500 rpm). The reaction was carried out under magnetic stirring for 3 h at room temperature (20 °C).Chitosan coating was performed as described previously ,33. A 7 −1 zinc acetate aqueous solution (MilliQ® water) was prepared at 25 °C. Then, 250 μL of this solution was added into 9.75 mL of chitosan-lecithin-polysorbate 80-coated lipid-core nanocapsule aqueous dispersion under magnetic stirring (500 rpm). After 1 min, 351.21 μL of this complexed nanocapsule aqueous dispersion was added of 648.79 μL of a 450 μg·mL−1 RGD aqueous solution under magnetic stirring (500 rpm) at room temperature (20 °C).The surface functionalization was carried out by forming an organometalic complex adapting the methodology previously described for nanocapsules decorated with an antibody fragment . A 1 mg·−1 phenylalanine aqueous solution under magnetic stirring (500 rpm), using a ligand/metal molar proportion of 3:1.In parallel, to obtain phenylalanine-functionalized nanocapsules, 250 μL of the zinc acetate solution was added into 9.75 mL of chitosan-lecithin-polysorbate 80-coated lipid-core nanocapsule aqueous dispersion under magnetic stirring (500 rpm). After 1 min, the reaction medium (10 mL) was added of 250 μL of 7.6 mg·mL−1 were respectively named RGD-MCMN (Dox100), RGD-MCMN (Dox500), Phe-MCMN (Dox100) and Phe-MCMN (Dox500).Formulations prepared without Dox were named RGD-MCMN and Phe-MCMN, while formulations containing Dox at 100 and 500 μg·mLFor the cell uptake study, fluorescent-labeled formulations were prepared using a PCL derivative as previously described by Poletto and co-workers . The con® ZS instrument . Mean diameter by intensity (z-average) was determined by PCS using the Cumulants method. The samples (20 μL) were diluted to 10 mL in purified water (1:500 v/v) and zeta potential was determined by laser Doppler micro-electrophoresis by Zetasizer Nanoseries® ZS instrument , after dilution of the samples in 10 mmol·L−1 NaCl aqueous solution (1:500 v/v).Photon correlation spectroscopy (PCS) and laser Doppler micro-electrophoresis were carried out in a Zetasizer Nanoseries−1), hydrodynamic diameter and median diameter by number of particles were determined by nanoparticle tracking analysis (NTA) in a NanoSight LM10 instrument  using the provided analytical software NTA 2.0 . This analysis allowed the observation of the Brownian motion recording the light scattered by the individual nanoparticles. Samples were diluted 5000× in ultrapure water  and injected into the sample chamber. Each video clip was captured over 60 s.The particle number density  were used as stationary phase and a solution of 0.1% trifluoroacetic acid and acetonitrile  at apparent pH 2.65, as the mobile phase. The flow rate was 1.0 mL·min−1, using an injection volume of 50 μL and detection at 254 nm.Dox was quantified by liquid chromatography (HPLC), adapting a method previously described . The sysEE%), the non-encapsulated fraction of DOX was also determined. The ultrafiltration-centrifugation technique was carried out using 10 kDa cutoff units (Merck KGaA) and centrifugation at 1844× g for 5 min . The non-encapsulated fraction of Dox (fraction of drug dissolved in the continuous phase) was determined by HPLC in the ultrafiltrate without dilution. Then, EE% was calculated using Equation (1).tC is the experimental drug content and Cnon-encap is the fraction of Dox dissolved in the water phase.To determine the experimental drug content , Dox was extracted from the formulations (0.5 mL) using acetonitrile (5 mL). The mixtures were stirred for 5 min and filtered  for injection (HPLC). To determine the encapsulation efficiency . To isolate the non-bound fraction of RGD, the RGD containing samples were placed in ultrafiltration-centrifugation units  centrifuged at 1840× g Relative Centrifugal Force (RCF) during 5 min . The ultrafiltrate was assay using the colorimetric method and the RGD concentration determined using the calibration curve. The yield of RGD complexation on the nanocapsule surface (Yield%) was determined using Equation (2).totalRGD is the RGD concentration in the formulation and non-boundRGD is the RGD concentration dissolved in the ultrafiltrate.The yield of RGD complexation on the nanocapsule surface was determined by an indirect method. The non-bound fraction of RGD in the formulations was quantified by a colorimetric method . According to the manufacturer’s instructions, a calibration curve was prepared using dilutions of bovine serum albumin (BSA) from 3 to 27 μg·mL2/95% air at 37 °C. Fresh medium was replaced every other day.Cell cultures were grown in DMEM (Dulbecco’s Modified Eagle) supplemented with 10% of fetal bovine serum (FBS), 0.1% amphotericin, 1% penicillin/streptomycin  in an atmosphere of 5% CO3 and 20.0 × 103 cells per well, respectively, in 24-well plates. After reaching 60% to 70% of confluency, cells were treated with the fluorescent-labeled RGD-MCMNf or fluorescent-labeled Phe-MCMNf using 5.15 × 10−3 μmol of particles per liter of well. The volume per well was completed to 500 μL with DMEM. After incubation for 24 h at 37 °C, formulations were removed and cells were washed with PBS (0.5 mL). After the second addition of PBS (0.5 mL), photomicrographies were obtained at 20× magnification with an Olympus IX71 fluorescent microscope  equipped with the CellSens Standard software .For confocal microscopy analysis cellular uptake, MCF-7 and U87MG cells were seeded at densities of 6.0 × 103 and 20.0 × 103 cells per well, respectively, in 24-well plates. After reaching 60% to 70% of confluency, cells were treated with the fluorescent-labeled RGD-MCMNf or fluorescent-labeled Phe-MCMNf using the concentrations of nanocapsules ranged from 1.03 × 10−4 to 5.15 × 10−3 μmol of particles per liter of well. The volume per well was completed to 500 μL with DMEM. After incubation for 24 h at 37 °C, formulations were removed and cells were gently washed with PBS buffer (3 times), detached with trypsin-EDTA, harvested and analyzed in a flow cytometer BD FACSVerse System  equipped with a PE filter in order to determine fluorescence intensity emitted by the fluorescent-labeled RGD-MCMN or fluorescent-labeled Phe-MCMN.Quantitative cellular uptake of fluorescent-labeled RGD-MCMN was evaluated by flow cytometry. MCF-7 and U87MG cells were seeded at densities of 6.0 × 10−4 to 5.15 × 10−3 μmol of particles per liter of well. The micromolar concentrations of nanocapsules were calculated using the Avogadro number (6.023 × 1023) considering the particle number density (PND) determined as described above using NTA. The cells were incubated for 24 and 72 h. After incubation, the medium was removed and each well was washed with phosphate-buffered saline to the addition of 0.5 mg·mL−1 MTT solution. The cells were incubated at 37 °C for 3 h. The MTT solution was removed and DMSO (100 μL) was added to dissolve the formazan crystals. Thus, the absorbance was measured at 560 nm and 630 nm on a microplate reader . The results were converted to cell viability, expressed as the percentage of cell viability against the control group, which did not receive any treatment (100% of cell viability).The 3--2,5-diphenyltetrazolium bromide (MTT) assay was used to estimate MCF-7 and U87MG cell viabilities. Cells were seeded 1500 cells per well for MCF-7 and 5000 cells per well for U87MG into 96-well plates. After 48 h, the medium (DMEM) was removed, the adherent cells were treated with each formulation and the volume completed to 100 μL with DMEM. The concentrations of nanocapsules in the treatments ranged from 1.03 × 10p < 0.05. For the cell viability results, the software MINITAB version 16.0  was used to plot the Pareto chart. A graphical display of the effect by factor was given in a Pareto chart, which analyzes the magnitude and the importance of each variable effect. The length of the bars in the chart is proportional to the effect. A factor was considered as “statistically significant” if its standardized effect exceeded a threshold. A line in the Pareto chart indicated the threshold for a test at the p-value of 0.05.All data are expressed as mean values ± standard deviation calculated from at least three independent experiments. Statistical analyses were performed using GraphPad Prism 5.0 . The level of significance was set to vβ3 integrin targeted antiangiogenic strategies for cancer treatment by intravenous administration.RGD surface modified LNC were successfully developed. Therefore, different Dox concentrations were nanoencapsulated into these nanocapsules. The cellular uptake of RGD-MCMN was significantly enhanced to the integrin-overexpressed cell lines (U87MG) compared to the integrin-deficient cell lines (MCF-7). Moreover, Pareto chart showed that the cell viability was mainly affected by Dox concentration and period of treatment in both MCF-7 and U87MG. The influence of RGD-functionalization on cell viability was a determinant factor exclusively to U87MG. We believe that this study provides a facile strategy towards the development of active-targeting drug-loaded nanocarriers for tumor therapy via integrin mediation. In this way, in vivo pre-clinical trials can be performed to evaluate the innovative formulations as a new chemotherapeutic strategy for drug delivery to tumor cells without damaging normal cells using α