Next, QHMF self-assembled into nano-dandelion, i.e. encapsulated Cur and Bai in water. Critical experimental conditions in the preparation process of nano-dandelion that could affect its final properties were explored. Nano-dandelion with a small particle size (121.0 ± 15 nm) and good normal distribution (PI = 0.129) could easily enter tumor tissue through vascular barrier. In addition, nano-dandelion with a suitable surface potential (–20.33 ± 4.02 mV) could remain stable for a long duration. Furthermore, good cellular penetration and tumor cytotoxicity of nano-dandelion were demonstrated through in vitro cellular studies. Finally, effective antitumor activity and reduced side effects were confirmed through in vivo antitumor experiments in A549 tumor-bearing nude mice.In this study, the novel carrier materials were screened to structure targeting nano-micelles (named ‘nano-dandelion’) for synchronous delivery of curcumin (Cur) and baicalin (Bai), which could effectively overcome the tumor resistance. Mannose (Man) was found to bind better to CD206 receptors on the surface of tumor-associated macrophages (TAMs), thereby increasing the number of nano-dandelion engulfed by TAMs. Furthermore, oligomeric hyaluronic acid (oHA) was able to target CD44 receptors, resulting in recruitment of a higher number of nano-dandelion to locate and engulf tumor cells. The disulfide bond (S–S) in 3,3′-dithiodipropionic acid (DA) could be broken by the high concentration of glutathione (GSH) in the tumor microenvironment (TME). Based on this, we selected DA to connect hydrophobic fragments  and oHA. A reduction-sensitive amphiphilic carrier material, quercetin–dithiodipropionic acid–oligomeric hyaluronic acid–mannose–ferulic acid  was fabricated and synthesized by  For example, lower pH , one of the essential immune cells, are involved in the recognition, phagocytosis, and killing of damaged tissues and potentially malignant cells  is used frequently as a biodegradable material in the field of nano-delivery systems because of its biocompatibility, high water solubility, and its ability to be easily chemically modified  has broad biological activity, including anti-inflammatory, antitumor, and anti-oxidation. Hence, Cur has been used by several researchers as an antitumor drug   was succin vitro release, cellular toxicity and uptake, TAM repolarization, in vivo distribution, and pharmacodynamics. The results suggested that combining therapy may be a promising therapeutic strategy for patients with NSCLC.As shown in 2.2.1.oHA (molecular weight <10 kDa) was obtained from Shandong Freda Co. Ltd. . Que, Man, DA, FA, Bai, formamide, tetrahydrofuran (THF), and 1-hydroxybenzotriazole hydrate (HOBT) were obtained from Aladdin Reagent Net . Dimethyl sulfoxide (DMSO) and petroleum ether were acquired from Tianjin Fuyu Chemical Industrial Corporation . GSH was purchased from Sigma-Aldrich . Dulbecco's minimum essential medium (DMEM) was obtained from Saiersi Biotechnology Co. Ltd. . Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co. Ltd. .2.2.2.2.1.The QHMF was synthesized in two parts: Que–S–S–oHA (QH) and Man-FA. The first part included the synthesis of QH. First, 126.40 mg (0.5 mM) of DA was dissolved in 5 mL of THF, and 120 µL (0.60 mM) of oxalyl chloride was added dropwise into the DA solution at 0 °C. Then, the solutions were mixed for 4 h (35 °C) under stirring and the solvent in the reaction solution was vaporized by rotary evaporators. A total of 153.33 mg (0.60 mM) Que was dissolved into 5 mL of THF, added to the reaction bottle, and the reaction was continued for 24 h at 42 °C under stirring. Finally, the Que–S–S was collected by rotary evaporator. The Que–S–S was dissolved in 3 mL of DMSO, and 1.2 equivalents of EDCI , and 1.2 equivalents of HOBT  were added and mixed at 45 °C under magnetic stirring. After 4 h, oHA  was dissolved in formamide (3.0 mL) and added dropwise into the Que–S–S solution and incubated at 52 °C with magnetic stirring for 48 h. Finally, the solvent was dislodged from the resulting solutions by dialyzing for 24 h at room temperature. The QH was obtained by lyophilization.FA–Man was synthesized via the EDCI/HOBT. FA  was dissolved in formamide (3.5 mL) at 60 °C. Then, 1.2 equivalents of EDCI  and 1.4 equivalents of HOBT  were dissolved in the formamide solution of FA. Next, the solutions were mixed at 45 °C for 4 h with magnetic stirring. Then, 1.2 equivalents of Man  were dissolved in formamide (2 mL) and then mixed into previous solutions. The mixtures were incubated at 55 °C for 36 h with magnetic stirring.The final step was the synthesis of QHMF. DMSO (6 mL) was used to dissolve QH, 1.4 equivalents of EDCI, and 1.6 equivalents of HOBT. Then, the solution was mixed at 42 °C for 5 h under stirring. Meanwhile, the formamide solution of FA–Man was added to the previous reaction bottle. The mixtures were incubated at 52 °C for 2 days with stirring. QHMF was obtained by dialysis and lyophilization.2.2.2.1H NMR was used to verify the structure of QHMF. QHMF (10.00 mg) was thoroughly dissolved in 0.6 mL of D2O and 0.3 mL of DMSO-d6, and the chemical shifts of QHMF were detected by 1H NMR.2.2.3.The nano-dandelion was prepared by the dialysis method. First, 10 mg of QH or QHMF was dissolved in 4 mL of formamide. Then, Cur (1.5 mg) and Bai (0.5 mg) were synchronously dissolved in formamide (2.0 mL). Next, two different solutions were mixed and transferred to a dialysis bag (2000 Da) and dialyzed in PBS (pH 7.4) for 12 h in the dark. Then, the unloaded Cur/Bai were removed from the residual solution by centrifuging at 2000 rpm for 10 min. The prepared nano-dandelion was obtained from the supernatant.2.2.4.The particle size and zeta potential of nano-dandelion were measured by Delsa Nano C . TEM was used to determine the morphological characteristics of nano-dandelion.Drug loading (DL) and entrapment efficiency (EE) were examined by HPLC   and then transferred into multiple dialysis bags (3000 Da). These dialysis bags were immersed in centrifuge tubes containing 45 mL PBS  with different concentrations of GSH . Subsequently, the tubes were incubated in a shaking (100 r/min) water bath at 37 °C. Release medium was sampled (1 mL) and the same amount of PBS was simultaneously added at the specified time point. The amount of Cur/Bai released was determined by HPLC at 425/278 nm.2.2.6.2.As CD44 and CD206 receptors are highly expressed on A549 and RAW264.7 cells, respectively, these two cell lines were used to evaluate the cellular levels of nano-dandelion. Both cell lines were cultured in DMEM supplemented with 10% FBS at 37 °C, 5% CO2.2.7.Concentration-dependence experiment. In this study, the fluorescence of Cur was used. A549 cells were cultured in 12-well plates for 12 h. Different concentrations  of free Cur, QH@Cur NMs, and QHMF@Cur NMs were added to the 12-well plates and incubated for 4 h. After washing and fixing, images were obtained with a fluorescent microscope  to monitor the cellular uptake of various NMs. Simultaneously, a concentration-dependence study on RAW264.7 was performed according to the above method using free Cur, QH@Cur NMs, and QHMF@Cur NMs.Time-dependence experiment. The time-dependence course of free Cur, QH@Cur NMs and QHMF@Cur NMs was determined in A549 or RAW264.7 cells. First, A549 (RAW264.7) cells were seeded in a 12-well plate. After 12 h, the cells were incubated with free Cur, QH@Cur NMs, and QHMF@Cur NMs  for the indicated time. After washing, cells were fixed with 4% paraformaldehyde for 15 min. Finally, images were obtained with a fluorescent microscope.Cell localization studies. Using Cur as a fluorescent probe and hochest33342 as a nuclear dye, the cellular localization of QHMF@Cur was determined using A549 (RAW264.7) cells seeded in six-well plates. Then, the cells were incubated with DMEM containing QHMF@Cur . The medium was removed after 4 h. After fixing and washing, 2 mL of hochest33342 (10 µg/mL) was used to stain cell nuclei. Finally, images showing cellular localization were obtained using a fluorescent microscope.2.2.8.In this study, we investigated the cytotoxicity of free Cur, free Bai, vacant NMs, and different NMs in A549 and RAW264.7 cells by MTT assay. Briefly, A549 and RAW264.7 (A549/RAW264.7) cells were simultaneously plated in 96-well plates and left for 12 h prior to treatment with the prepared free Cur, free Bai, vacant NMs, and different NMs.After incubating for 24 or 48 h, 20 μL of MTT solution (5 mg/mL) was dripped into 96-well plates and cells were incubated for 4 h. Then, the supernatant was carefully replaced by 200 mL of DMSO. Finally, the absorbance was measured using a microplate reader  at 570 nm after 10 min of oscillation. Cell viability  was calculated according to the following formula:2.2.9.Conditioned culture medium was collected from A549 cells. RAW264.7 cells were cultured in 96-well plates with collected culture medium. Then, the culture medium was removed, and the cells were treated with different concentrations of QHMF@Bai for 24 h. After collection and centrifugation, the levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the medium were measured by ELISA.2.2.10.In vivo fluorescence imaging studies were performed using A549 lung cancer-bearing mice. The mice were treated with 150 µL free DiR, QH@DiR, or QHMF@DiR via tail vein injection once the tumor had grown to an appropriate size. The distribution of fluorescence in vivo was monitored at set time points by In-Vivo FX Pro . Twelve hours after injection of DiR, tumor tissue and the major organs were harvested from mice, and fluorescence intensity of each organ was monitored using In-Vivo FX Pro.2.2.11.in vivo was investigated using ectopic A549 lung cancer-bearing mice, which were randomly divided into six groups. The antitumor efficacy of saline (control), free Cur, free Cur/Bai, QHMF@Cur, QH@Cur/Bai, and QHMF@Cur/Bai was evaluated in lung cancer-bearing mice following administration of multiple doses equivalent to 1 µg/g  via tail vein injection. The tumor volume, as a therapeutic index, and body weight of mice, as a measure of toxicity, were recorded at each time point. The change in tumor volume in mice was used as an indicator of therapeutic efficacy. Finally, the major organs and tumor should be collected and fixed in 4% paraformaldehyde solution immediately after the mice were humanely sacrificed. After paraffin-imbedding and slicing, the sections were stained with hematoxylin–eosin (H&E) dyes and images showing histopathological evaluation were obtained by using a fluorescent microscope.The antitumor activity of NMs The size of the tumor was determined using the followed equation:3.3.1.1H NMR spectra of QH and QHMF are shown in 2–S–S–CH2) and Que (the ‘H’ of benzene ring), respectively. Additionally, compared with QH, typical signal of FA was observed at 9.8 ppm  and concentration-dependent (b) study in A549 cells; Time-dependent (c) and concentration-dependent study (d) in RAW264.7 cells. Localization study in A549 and RAW264.7 cells (e).Furthermore, the QHMF NMs were mainly taken into the cytoplasm of A549 and RAW264.7 cells, as shown in 3.5.A549 cells overexpressing CD44 receptors and RAW264.7 cells overexpressing CD206 receptors were used in a cytotoxicity study. The inhibiting effects of free Cur, free Bai, free Cur/Bai, QHMF@Cur, QHMF@Bai, and QHMF@Cur/Bai on the A549/RAW264.7 cells were determined by the MTT assay .3.6.Bai had been reported to have the ability to reprogram TAMs toward an M1-like phenotype from M2-like phenotype. Next, to examine whether Bai could reprogram macrophages toward an M1-like phenotype, leading to tumor suppression, we measured the presence of IL-6 and TNF-α in the supernatant of RAW264.7 cells following treatment with Bai . High co3.7.in vivo, DiR-labeled QH NMs and QHMF NMs were injected into A549 tumor-bearing nude mice, and in vivo real-time fluorescence imaging is shown in To estimate the biodistribution of QH NMs and QHMF NMs Interestingly, QH@DiR NMs and QHMF@DiR NMs readily accumulated in tumors compared with free DiR, especially the QHMF@DiR NMs. Fluorescent imaging of isolated tissues  showed t3.8.We evaluated the antitumor effects of saline (control), free Cur, free Cur/Bai, QHMF@Cur, QH@Cur/Bai, and QHMF@Cur/Bai .First, changes in the body weights of mice are shown in 3.9.The toxicity levels of various formulations were measured in tumors and major organs of mice. Histological analysis of tumor and major organs is shown in 4.in vitro cytotoxicity test) and tumor inhibition rate (in vivo pharmacodynamics experiment). Remodeling of TAMs had been successfully verified by detecting changes in the secretion of IL-6 and TNF-α secreted from TAMs and we realized that two sets of data were not abundant. We will study the specific proteins of M1 and M2 in the next experiment, which further proved the successful transformation of TAM. Besides, we found that baicalin could promote TAMs to secrete more IL-6 and TNF-α, but baicalein was not. It was not hard to speculate that β-D-glucuronide was an indispensable part for Bai when Bai plays a remodeling role. In the next following study, we will conduct more in-depth research, including the mechanism that Bai promotes the transformation of TAM, etc.In this work, we demonstrated that Cur/Bai-based combination therapy had the better anti-cancer effects than individual Cur application, such as better A549 cells inhibition rate . The DiR remains in the tail of the mouse and it was not cleaned up in time.5.1H NMR. Analysis of particle size and morphology showed that nano-dandelion presented a uniformly distributed spherical structure of smaller size. Furthermore, nano-dandelion presented a suitable surface charge, which could be stable in systemic circulation. The reduction-sensitivity of nano-dandelion was demonstrated by an in vitro release assay. In vitro cell experiments showed that improved cellular uptake and cytotoxicity were achieved by nano-dandelion. Additionally, TAMs were successfully transformed into tumor-killing M1-type macrophages via reprograming by Bai, demonstrating promising role as a tumor adjuvant therapy. In vivo studies have shown that nano-dandelion could readily aggregate at tumor sites and exert more effective tumor-suppressive effect compared to free drugs. Furthermore, rate of tumor inhibition with QHMF@Cur/Bai NMs was higher than that with QHMF@Cur NMs, which further confirmed that Bai exerted an effective adjuvant antitumor effect. Overall, our proposed adjuvant tumor therapy based on reprograming of TAMs represents a promising strategy with good research prospects.To further enhance the efficacy of Cur and to avoid development of multidrug resistance by tumor cells, we introduced Bai as an adjuvant therapy to reprogram TAMs. In this study, we designed a novel amphiphilic nanocarrier material (QHMF) with reduction-sensitive potential using few simple chemical reactions to self-assemble a micellar delivery system, nano-dandelion. We demonstrated that QHMF was successfully synthesized by


ACS94 rapidly enters the cells, where it is metabolised to cysteine and the dithiolethione moiety ACS48. Experiments performed through the oral administration of ACS94 to healthy rats showed that it is capable of increasing the GSH levels in most of the analysed organs and the concentration of circulating H2S. Although the increase in GSH concentration was similar to that obtained by ACS48 and N-acetylcysteine ethyl ester, the H2S increase was long-lasting and more evident with respect to the parent molecules. Moreover, a decrease of homocysteine in several rat organs and in plasma was noted. This effect may represent a potential therapeutic use of ACS94, as hyperhomocysteinaemia is considered a risk factor for cardiovascular diseases. Lastly, ACS94 was more efficient than N-acetylcysteine in protecting the liver and kidneys against acute acetaminophen toxicity.The synthesis of a new dithiolethione-cysteine ethyl ester hybrid, ACS94, its metabolites, and its effect on GSH levels in rat tissues and on the concentration of circulating H  Dithiolethiones are a class of sulphur-containing molecule, primarily of synthetic origin, but also found in trace amounts in certain cruciferous vegetables. They have been established as anticarcinogens and protective compounds against radiation injury and hepatotoxicity induced by carbon tetrachloride or acetaminophen (APAP)2S donors under physiological conditionsH-1,2-dithiole-3-thione (ADTOH) into existing drugs,in vitro tests for these compounds suggest that, in addition to maintaining the parent molecule’s therapeutic capacities, they also have several other advantages   7. The ,2S and GSH content in several rat organs.GSH plays an important role in the detoxification of xenobiotics, of their metabolites, and of reactive oxygen species (ROS), and would, therefore, be attractive for increasing its availability in man. As Cys concentration in cells is limiting to GSH synthesis, supplementing Cys or its precursors appears to be a logical choice for boosting its levels in cells. Cys is generally not used because it is rapidly oxidised to the insoluble cystine form, thus making the preparation of stable formulations difficult. Furthermore, it has been reported that Cys at high concentrations is toxic for cultured cells, and its content is therefore usually kept low in parenteral formulationsH-1,2-dithiol-4-yl)benzoic acid (ACS48) to the amino group of cysteine ethyl ester  equipped with a non-reverse probe at 25° C, using DMSO-d6 as a solvent. The chemical shifts were expressed in ppm (δ), coupling constants (J) in Hertz (Hz). The high-resolution mass spectra (HRMS) were performed with an Apex II ICR-FTMS Bruker Daltonics mass spectrometer  in positive or negative electrospray ionization (ESI).Unless otherwise indicated, all the chemicals and solvents required for the syntheses and the analyses were of high purity grade and were purchased from Sigma-Aldrich . CC = flash column chromatography. Melting points (m.p.) were determined in open capillary tubes with a Büchi apparatus  and were uncorrected. N-acetylcysteine ethyl ester (NACET)H-1,2-dithiol-4-yl)benzoic acid (ACS48)The S)-trityl-L-cysteine ethyl esterH-1,2-dithiol-4-yl)benzoic acid , 1-hydroxybenzotriazole , and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride  in anhydrous DMF (3 ml). The mixture was stirred for 4 h at r.t. under N2. Following the evaporation of the solvent under reduced pressure, the residue was taken up with CH2Cl2 and the organic phase was washed with water, dried over anhydrous Na2SO4, and evaporated to dryness. The crude compound was then purified by CC , yielding pure 2-benzamido)-3-(tritylthio)propanoic acid ethyl ester . M.p. 86–93 °C. 1H-NMR (DMSO-d6): δ = 9.22 ; 8.96 ; 7.90 ; 7.68 ; 7.35–7.22 ; 4.25–4.17 ; 4.02–3.95 ; 3.31 ; 2.83–2.71 ; 1.09–1.04 .A solution of benzamido)-3-(tritylthio)propanoic acid ethyl ester  in 4 N HCl in dioxane (10 ml) at 0 °C; water (1 ml) was added, and the mixture was stirred at 0 °C for 30 min. The solvent was evaporated to dryness, and the residue was taken up with ether and then evaporated. This procedure was repeated several times to ensure the complete elimination of HCl and dioxane. After rinsing the residue, first with CH2Cl2 and then with ether, pure ACS94  was obtained. M.p. 109–111 °C. 1H-NMR (DMSO-d6): δ = 9.22 ; 8.84 ; 7.93 ; 7.68  4.56–4.50 ; 4.11 ; 2.99–2.83 ; 2.68 ; 1.18 . 13C-NMR : δ = 214.0, 170.8, 166.7, 160.4, 137.0, 133.0, 127.8, 129.3, 61.1, 56.1, 25.5, 14.5. HRMS (ESI) m/z calcd for C15H15NO3S4Na [M + Na]+: 407.98270; found: 407.98251.The trityl deprotection of the thiol was performed by dissolving 2- in anhydrous DMF (3 ml) was added drop wise to a solution of ACS48 , HOBt , and EDAC  in anhydrous DMF (4 ml). The mixture was stirred at r.t. for 24 h, under N2. After the solvent was evaporated to dryness, the crude residue was taken up with CH2Cl2 and the organic solution was washed with water, dried over anhydrous Na2SO4, and evaporated to dryness. The crude compound was then purified by CC . After rinsing with ether, pure ACS96 was obtained . M.p. 98–102 °C.A solution of L-cystine ethyl ester dihydrochloride1H-NMR: (DMSO-d6) δ = 9.18 ; 8.97 ; 7.87 ; 7.65 ; 4.77–4.70 ; 4.16–4.09 ; 3.31–3.09 ; 1.20–1.16 . 13C-NMR : δ = 214.0, 171.0, 166.6, 160.4, 147.5, 137.0, 133.8, 129.3, 127.7, 61.5, 52.5, 14.5. HRMS (ESI) m/z calcd for C30H28N2O6S8Na [M + Na]+: 790.96052; found: 790.96138.tertbutyl ester2. After the solvent was evaporated to dryness, the crude residue was taken up with CH2Cl2, and the organic solution was washed with water, dried over anhydrous Na2SO4, and evaporated to dryness. The crude compound was then purified by CC . After rinsing with ether, pure tertbutyl 3,3′-disulfanediylbisbenzamido)propanoate) was obtained . M.p. 88–90 °C. 1H-NMR (DMSO-d6): δ = 9.18 ; 8.97 ; 7.87 ; 7.65 ; 4.77–4.70 ; 3.31–3.09 ; 1.48 .A solution of cystine tertbutyl 3,3′-disulfanediylbisbenzamido)propanoate)  in 4 N HCl in dioxane (30 ml) at 0 °C as a scavenger, and the mixture was stirred at r.t. for 3 h. The solvent was evaporated to dryness and the residue was taken up with ether and then evaporated. This procedure was repeated several times to ensure the complete elimination of HCl and dioxane. After rinsing the residue, first with CH2Cl2 and then with ether, pure ACS97  was obtained. M.p. 254–255 °C. 1H-NMR (DMSO-d6): δ = 9.19 ; 8.85 ; 7.87 ; 7.64 ; 4.73–4.67 ; 3.55–3.07 . 13C-NMR : δ = 214.0, 172.4, 166.5, 160.2, 147.5, 136.9, 134.0, 129.2, 127.6, 52.5. HRMS (ESI) m/z calcd for C26H19N2O6S8 [M–H]–: 710.90143; found: 710.90386.Water (5 ml) was added to a solution of ad libitum for 2–3 weeks before their use and during the experiments. All animal handling procedures were carried out in accordance with the European Community guidelines for the use of laboratory animals. The experiments were authorised by the local ethical committee of the University of Siena.Sprague–Dawley male rats (300 g) were purchased from Charles River . The rats were kept under controlled conditions  and fed The rats were implanted with either a single or double valve . The jugular and femoral veins were cannulated  for compound administration  and blood collection (jugular), as previously described3EDTA, and were immediately centrifuged at 10,000×g for 20 s to obtain plasma.The rats received intravenous (iv) administrations of ACS94 (20 mg/kg) through the femoral vein. At the indicated times, blood aliquots (200 μl each) were collected through the valve connected to the jugular vein in tubes containing 5 μl 50 mg/ml Kv/v) pH 3.09; solvent B = acetonitrile; 0–5 min: 94% solvent A/6% solvent B; 5–10 min linear gradient from 6% to 10% solvent B, 10–10.5 min linear gradient from 10% to 14% solvent B, 10.5–14.5 min 14% solvent B, 14.5–15 min linear gradient from 14% to 25% solvent B, 15–19 min linear gradient from 25% to 33% solvent B. A constant flow rate of 1.2 ml/min was applied. Detection was performed at 390 nm excitation and at 480 nm wavelength emission. Asymmetrical disulphide with Cys (ACS94-Cys) was produced by reacting 0.2 M ACS94 with 0.1 M cystine in a 10 mM phosphate buffer pH 8.0 for 2 days at room temperature. The identities of the peaks were analysed by adding the standard compounds, by analysing the same samples pretreated with dithiotreithol (DTT), and by comparing the retention times of the different peaks. One aliquot of plasma (20 μl) was used for H2S analysis.The analysis of the ACS94 metabolites in the plasma was carried out by deproteinising one aliquot of plasma (50 μl) through the addition of three volumes of acetonitrile. The supernatants were then acidified through the addition of trifluoroacetic acid and were loaded into HPLC. The HPLC separation was performed on a C18 column . Elution conditions: solvent A = sodium acetate 0.25%  and organs were rapidly removed, washed in NaCl 0.9% (w/v) and then immediately frozen in liquid nitrogen and stored at –80 °C until analyses. Aliquots of blood (0.4 ml) were rapidly centrifuged at 10,000×g for 20 s to obtain plasma. Plasma was stored at –20 °C and used for the analysis of metabolites and total thiols. Erythrocytes were washed in Na+/K+ phosphate buffered saline containing 5 mM glucose and then haemolysed by the addition of 100 volumes of 0.02 M Na+/K+ phosphate buffer. One aliquot of frozen tissues was homogenised  by Teflon/glass potter in acetonitrile for the analysis of ACS94 and its metabolites (see procedure described above); one other aliquot was homogenised  in a 20% (w/v) TCA solution containing 1 mM K3EDTA and 0.2 mM diethylenetriaminepentaacetic acid (DTPA) for H2S detection. A third aliquot of frozen tissues was used for thiol analysis.For the study of drug distribution, animals were treated with endovenous infusion through the femoral vein with a solution of 20 mg/kg ACS94 or vehicle. After 60 min of the treatment rats were killed by decapitation, blood (about 2 ml) was collected in EDTA-containing tubes , the rats’ blood and organs were rapidly removed under anaesthesia . Plasma was obtained through the centrifugation of whole blood at 10,000×g for 20 s. One aliquot (20 μl) was used for the fresh analysis of H2S, whereas the rest of the plasma was stored at –80 °C for the analysis of the total thiols. The organs were washed in NaCl 0.9% (w/v) and then immediately frozen in liquid nitrogen and stored at –80° until the time of analyses. The GSH was measured in the organ homogenates through HPLC after conjugation with monobromobimane (mBrB), as previously describedAnimals implanted with a single valve for blood collection were treated orally (by gavage) with 10 mg/kg twice a day of ACS94 or equimolar NAC, NACET, ACS48 for four days. Aliquots of blood (0.2 ml) were collected every 12 h for H2S was detected through a modification of the methylene blue methodw/v) TCA solution containing 1 mM K3EDTA and 0.2 mM DTPA. After 30 s of centrifugation at 10,000×g, 40 μl of the supernatant were added with 5 μl of N,N-dimethyl-p-phenylenediamine sulphate , followed by 5 μl of FeCl3 (30 mM in HCl 1.2 N); after 20′ of incubation in the dark, the samples were separated through HPLC using a Zorbax Eclipse XDB-C18 column . The methylene blue formed from the reaction of H2S with DPD in the presence of FeCl3 was monitored at a wavelength of 667 nm using a diode array detector.Hw/v) sodium dodecyl sulphate, and were put in a shaker until complete dissolution. Aliquots of the samples were then diluted with the same buffer and reacted with an excess of DTNB, as previously describedThe animals were randomly divided into four groups, each consisting of four animals. APAP was dissolved in saline and injected ip at the dose of 2 g/kg. The group 1 rats served as a control group, and received a single dose ip injection of 1 ml saline containing 10% DMSO and 0.5% carboxymethylcellulose. The group 2 rats were treated with a single dose of APAP + vehicle. The group 3 and 4 rats received APAP + ACS94 (50 mg/kg) or equimolar NAC. The research drugs, dissolved in saline containing 10% DMSO and 0.5% carboxymethylcellulose, were administered orally 15 min before and 2 and 4 h after APAP administration. Six hours after the APAP treatment, the rats were anaesthetised with pentobarbital (60 mg/kg), after which blood from the abdominal aorta and organs were collected for biochemical analyses. After conjugation with mBrB, as above described, the GSH and Hcys were measured in the tissue homogenates through HPLC. After the pretreatment of the samples with DTT, the total Hcys was measured in the plasma through HPLC and mBrB labelling. The alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, uric acid, urea, and creatinine in the plasma were measured using the Roche COBAS 6000 instrument (Roche Diagnostics S.p.A. Monza (MB), Italy). The protein SH groups (PSH) were determined in aliquots of TCA deproteinised samples: specifically, the protein pellets were re-suspended with a glass rod in a 0.2 M phosphate buffer, pH 7.4, containing 2% , Italy). equipped with diode array and a fluorimetric detector was used for all determinations. All the spectrophotometric determinations were carried out using a Jasco V/550 instrument , Italy).p < .05 was considered statistically significant.The data are expressed as means ± SD. The differences between the means were evaluated using one-way analysis of variance (ANOVA) with the Newman–Keuls multiple test, as appropriate. A value of H-1,2-dithiol-4-yl)benzoic acid (ACS48)tertbutyl ester3-Mercapto-2-benzamido)propanoic acid ethyl ester (ACS94) and its two main metabolites diethyl 3,3′-disulfanediylbisbenzamido)propanoate), ACS96 and 3,3′-disulfanediylbisbenzamido)propanoic acid) ACS97 have been synthesised, respectively, by coupling 4- but we did not find any significant alteration induced by ACS94 (data not shown).2S production from cells, in vivo, can be stimulated primarily by increasing cellular stores of cysteine, which can function as a substrate of cystathionine γ-lyase (CSE). We, therefore, compared the circulating levels of H2S after the iv or oral administration of ACS94 with those obtained after treatment with equimolar doses of NAC, NACET, or ACS48 , which causes hepatic damage. It has been suggested that NAPQI reacts with free thiol groups of proteins and, in turn, leads to mitochondrial damage and nuclear DNA fragmentationAcetaminophen metabolism is known to involve cytochrome P450 to form p < .01). This decrease was not able to be observed in the NAC-treated animals. In addition, most of the indicators of liver/kidney damage were consistently lower in the ACS94 treated animals with respect to those treated with NAC  may accumulate, leading to protein ,A decrease in GSH has been linked to the onset and/or progression of many diseases, and there is consequently considerable interest in counteracting this phenomenon. N-acetylcysteine has been widely studied as a GSH enhancer, as it is considered to be a source of Cys. However, despite the fact that it has been tested for treating numerous diseases, up until now it has only been used for paracetamol intoxication, since, in most cases, it did not work to ameliorate the symptoms2S biology is quite complex, interest in it is growing. In fact, even if H2S is known to be toxic for humans by targeting cytochrome-c oxidase, there is much evidence regarding its protective role in several disease states, with these different effects being related to its levels. At low physiological concentrations, H2S can act as a modulator of numerous biochemical pathways, thus providing beneficial effects. Evidence of this is available for the nervous, gastrointestinal, cardiovascular, and respiratory systems2S to target tissues. These essentially fall into three categories: sulphide salts, naturally occurring compounds, and synthetic H2S donorsAlthough H2S in plasma2S, such as S-propylcysteine, S-allylcysteine, S-propargylcysteine2S levels did not increase in the NAC-treated animals.We have recently studied two different synthetic compounds that provided promising results in experiments with rats. One of these is NACET, the ethyl ester of NAC. Chronic treatments with NACET in comparison with equimolar NAC indicated that only NACET was capable of increasing GSH significantly in most of the analysed organs. Furthermore, only NACET was able to cross the blood brain barrier, thus increasing the GSH in the brain. We found that the pharmacokinetic profile of this molecule is peculiar, in that it is readily absorbed and its bioavailability is quite high (more than 60%), whereas the bioavailability of NAC is below 5%. NACET was also able to increase the physiological levels of H2S levels in plasma,,,per se capable of increasing GSH levels. It has been hypothesised that several of the antioxidant enzymes involved in GSH synthesis/metabolism, such as γ-glutamylcysteine synthase, GSH transferase, and GSH reductase, may be altered by these drugs,2S levels is still unknown, in our experiments we were able to observe a biphasic increase in the same, which can be explained by admitting the involvement of two different pathways.Dithiolethiones were also shown to be effective at increasing GSH levels in several rat organs, including the brain, and H2S levels in rat organs and plasma, respectively  with cysteine ethyl ester. As hypothesised, it maintained the ability to increase both GSH and Hectively . ACS94 w reached . The dit,The metabolism of ACS94 is quite complex, as the molecule can undergo: de-esterification reactions, hydrolysis reactions, and thiol oxidation to disulphide by thiol-disulphide exchange reactions. As a consequence of this, many metabolites can form  and 3. T2S production.However, after 60 min from the treatment with ACS94, we could observe a higher concentration of its de-esterified forms within cells of all the studied rat tissues with respect to the plasma. It is probable that, in analogy to NACET, the fraction of ACS94 that is de-esterified is trapped within cells, as it becomes more hydrophilic, and, consequently, its exit is restricted. Within the cells, it is then hydrolysed mainly to cysteine and the dithiolethione moiety. Both cysteine and ACS48 are efficient GSH enhancers , which is known to be redox regulated2S could be due to both enhanced TSP and increased Cys levels. This might also explain the biphasic release of H2S from the dithiolethiones: the initial increase (observed immediately after the drug’s administration) is likely due to its direct release from the dithiolethione-containing drugs, whereas the later increase (6–24 h after the treatments) may be attributable to the activation of TSP and generation from Cys. Moreover, our previous data indicate that dithiolethione treatments are followed by enhanced Cys and GSH release from the intracellular compartment to the blood, thus leading to an increase in the thiol to disulphide ratio in the plasma. Considering that Hcys mainly occurs in plasma as a disulphide (both with proteins and with low molecular mass thiols), the thiols occurring in the plasma can react with these disulphide forms to release free Hcys, which, in turn, can be metabolised. NACET was also able to decrease Hcys, and, like the Cys prodrug, we can hypothesise that it may promote a decrease in Hcys levels by reducing the disulphide forms of Hcys itself in the extracellular milieu, but without any involvement of the TSP.The evidence that ACS94 also maintained the ability to lower Hcys in plasma   may be o,Another pharmacological effect that ACS94 may exert is that of liver protection against intoxication with APAP. As a supplier of GSH, NAC is regarded as the antidote of choice for treating acetaminophen overdose. However, under our experimental conditions, NAC was only shown to exert some degree of protection against kidney toxicity . Along this time, we could not observe any change in the rat weight for every kind of administered drug . Further long-lasting experiments, with targeted experiments and dose–response studies are necessary to characterise the toxic potential of ACS94.2S in vivo, and this effect is long-lasting; 3) it is capable of providing antioxidant protection by increasing GSH and by possessing an –SH group itself; 4) it significantly decreases Hcys levels in several rat target organs and in plasma; 5) it prevents APAP induced thiol depletion in rat kidneys and liver. ACS94 can affect the metabolic pathways relating to Hcys, Cys, GSH (by TSP pathway), and H2S. The dysregulation of these pathways is associated with several pathologies, mostly at a cardiovascular level2S and GSH, ACS94 is potentially capable of protect against these disorders. Moreover, thanks to its antioxidant properties, it can have beneficial effects by protecting the cells against ROS overproduction, which mostly occurs in inflammatory diseases. Considering that acetaminophen poisoning currently represents the most common cause of ALF in North America and EuropeIn this article we have proposed a new synthetic compound that possesses several promising pharmacological properties: 1) it has good pharmacokinetics, as it rapidly enters the cells; 2) it releases H


Mycobacterium agri and Gordonia cholesterolivorans were identified as most potent applying a new “reverse discovery” approach. This relies on the possibility that efficient strains that are already isolated and deposited can be identified through enrichment cultures. These strains formed massive biofilms on the octocrylene droplets. GC-MS analysis after cultivation for 10 days with M. agri revealed a decrease in octocrylene concentration of 19.1%. LC-MS/MS analysis was utilized in the detection and quantification of transformation products of octocrylene. M. agri thus represents an ideal candidate for bioremediation studies with octocrylene and related compounds.Octocrylene is a widely used synthetic UV absorber of sunscreens and found in several environments. Ecological consequences of the accumulation of UV filters are widely discussed. This is the first report revealing the microbial potential to transform octocrylene. A microbial community comprising four bacterial species was enriched from a landfill site using octocrylene as carbon source. From these microorganisms  Mycobacterium agri and Gordonia cholesterolivorans) can form biofilms on its surface. They also report that cultivation for 10 days with Mycobacterium agri decreases octocrylene concentration by 19%.Suleiman et al. use “reverse discovery” approach to identify four bacterial species that were enriched on the UV filter octocrylene, two of which ( Recently, several studies focused on the extensive use and potential ecological consequences of octocrylene, since the compound was detected in various water and sediment samples in wastewater2 oceans3, lakes, and rivers5. Moreover, octocrylene was found in the liver tissue of Franciscana dolphins and accumulation of octocrylene in zebrafish was reported upon exposure7. Further, octocrylene was suggested to potentially affect transcription of genes in the zebrafish’s brain and liver9. So far, little is known about potential degradation and biotransformation of octocrylene by microorganisms. Hence, this is the first study, to our knowledge, which reports on microbial growth in the presence of octocrylene and its degradation and transformation to other compounds.Octocrylene  is a synthetic organic filter widely used as UV-absorber (in the UVB region) in sunscreens and personal care products10. The microbial community of the OC-colonizing enrichment culture consisted of four genera, and the distinct bands of the DGGE were obtained and assigned to the bacterial genera Gordonia (100% identity), Mycobacterium (100% identity), and Hydrogenophaga (99% identity) after excision of the respective bands and sequencing . Samples were subsequently used for enrichment cultures (inoculation 1% (v/v)) with 0.35% (v/v) octocrylene as carbon source. After 14 days, grown cultures were transferred into fresh medium with two subsequent streakings. Repeatedly, biomass formation on the octocrylene droplet surface could be observed and the medium became turbid compared to the controls. In order to investigate the community composition of the culture, DGGE analysis was performedMycobacterium and Gordonia species have been reported to be associated with degradation of polluting polycyclic aromatic hydrocarbons (PAHs), such as pyrene or oil, and were proposed for application in environmental bioremediation14. Although different techniques were performed, isolation of pure strains from the enrichment culture was not possible and resulted in mixed cultures. Therefore, the “reverse discovery” approach was integrated. This approach is based on taking use of already isolated and deposited pure strains, showing high identities to the identified 16S rRNA genes amplified from the organisms of the enrichment culture. Highly identical strains can be purchased from culture collections, in order to identify the most efficient strains capable of degrading octocrylene. Likewise, several deposited strains at the German Culture Collection (DSMZ) belonging to the genera Mycobacterium and Gordonia, whose 16S rRNA gene showed high identities to the DGGE-identified ones, were tested for their ability to grow in modified DSM media 645 and 65 at 37 °C and 30 °C in the presence of 0.35% (v/v) octocrylene. Interestingly, M. agri (DSM 44515) and Gordonia cholesterolivorans (DSM 45229) were able to colonize the octocrylene droplet rapidly forming massive biofilms on its surface . Interestingly, in absence of external carbon sources, no biofilms were formed at all, indicating the necessity of small amounts of accessible energy sources to colonize the octocrylene droplet.M. agri and G. cholesterolivorans, gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses were performed with the pure cultures grown with 0.35% octocrylene (v/v). Control experiments were conducted with M. agri and G. cholesterolivorans grown in the absence and presence of octocrylene. In addition, the medium containing octocrylene was incubated under the same conditions without inoculation. After 10 days of cultivation, triplicates of cultures and controls were used for GC-MS and LC-MS/MS analyses. Samples were prepared by extraction of the culture using hexane/dichloromethane (1 + 2), for LC-MS/MS measurements the GC extract was evaporated to dryness and refilled with acetonitrile, and the amount of octocrylene was determined using the GC-MS-SIM mode, with m/z 249 as the quantifying ion. The qualitative recording of products (product scan) was done by GC-MS-TIC mode. Separation was performed on an Agilent DB5MS ultra inert capillary column.Hence, in order to study transformation of octocrylene by M. agri, the concentration of octocrylene measured by GC-MS-SIM mode decreased from 3536 ± 239 mg/L (controls) to a final concentration of 2861 ± 193 mg/L at day 10, representing a decrease of 19.1% octocrylene butyl-2-cyano-3,3-diphenylacrylate (DOCCA), 2-Cyano-3,3-diphenylacrylic-acid (CPAA), and 2-Ethyl-5-hydroxyhexyl-2-cyano-3,3-diphenylacrylate (5OH-OC) were synthesized and provided by Bury et al.9. Consequently, we were able to compare GC-MS spectra obtained in this work with the metabolite’s GC-MS spectra and LC-MS/MS which was subsequently used to quantify the metabolites in our experimental design. While the metabolites DOCCA, CPAA, and 5OH-OC were identified in the MS spectra and quantified via LC-MS/MS, one occurring peak in GC-MS run  were further metabolized by the microorganism and utilized for growth. Non-biotic factors responsible for the reduction of octocrylene can be excluded, since controls without M. agri did not show any decrease in octocrylene concentration.LC-MS/MS revealed the occurrence of DOCCA with 165 ± 60 µg/L, of CPAA with 11.8 ± 2.6 µg/L and of 5OH-OC with 246.0 ± 34 µg/L. This indicates that G. cholesterolivorans revealed no change of octocrylene concentration during incubation, although biofilm was observed. This indicates that in this case biofilm formation does not go automatically along with transformation or degradation of octocrylene.Interestingly, the analysis of growth experiments with 3 and solutions for this pollution have to be investigated. Man-made biofilms were proposed previously for bioremediation of sewage effluent contaminated with hydrocarbons16. M. agri represents a potential candidate for bioremediation, since it belongs to the rapidly growing Mycobacteria and can be found ubiquitously in soil and water systems18. Therefore, a novel eco-friendly application using M. agri or specific microbial consortia could be investigated and applied for degradation of chemical UV filter like octocrylene. This work highlights the potential of heterotrophic microorganisms to transform octocrylene in lab experiments. Due to the omnipresence of M. agri in the environment, it is conceivable that M. agri will also be able to transform the UV filter octocrylene in nature. Hence, further analysis should be conducted regarding biotransformation of octocrylene by microorganisms in natural sediments and waters. Future studies will reveal the metabolic pathway for octocrylene degradation and the key enzymes involved in this process.Since UV filters such as octocrylene accumulate in nature, the identification of further octocrylene degrading microorganisms is desirable. It has been reported that marine environments are endangered by chemicals and UV-filters of sunscreen4, 0.1 g/L MgCl2, 0.5 g/L NH4SO4, 0.5 g NH4NO3, 0.1 g/L CaCl2, 40 mM phosphate buffer, trace element and vitamin solution 141, and 0.005% (w/v) yeast extract). Incubation bottles were inoculated with 1% (w/v) of the soil sample and supplemented with 0.35% (v/v) octocrylene as carbon source. After 14 days of incubation, grown cultures were transferred into fresh medium with two subsequent streakings.Sediment and water samples were taken from an on-site sewage plant and soakage at a landfill site in Singhofen (Germany). The samples were subsequently used for enrichment cultures (volume 20 mL) using oxic medium .10 DGGE was performed by employing a DcodeTM System . A 6% polyacrylamide gel with a denaturing gradient of 35–70% formamide was used and electrophoresis ran for 17 h, at 55 °C and 100 V. The gel was stained in Roti-Gel Stain  and afterwards analyzed with a UV transilluminator. In addition, selected bands were cut out and DNA was extracted from the gel by incubation in 10 µL sterile water over night at 4 °C, amplified by PCR and amplificated were sequenced (Eurofins Genomics).DGGE analysis was performed as described previously.Mycobacterium sp. and Gordonia sp. showing within their 16S rRNA genes high similarities compared to the sequenced and identified fragments of the DGGE analysis were chosen and ordered from the DSMZ. Mycobacterium agri (DSM 44515), M. aichiense (DSM 44147) and M. aromaticivorans (DSM 45407) were incubated in DSMZ medium 645 2SO4, 1.5 g KH2PO4, 1.5 g Na2HPO4, 0.4 g Na-citrate, 0.025 g MgSO4, 0.5 mg CaCl2, 1 mg ZnSO4, 1 mg CuSO4, 0.5 g L-glutamic acid, 0.04 g ferric ammonium citrate, 1 mg pyridoxine, 0.5 mg biotin, 0.05 g oleic acid, 0.85 NaCl, 5 g BSA, 2 g glucose) supplemented with 0.35% octocrylene at a temperature of 37 °C. Gordonia caeni (DSM 45852), G. cholesterolivorans (DSM 45229), and G. defluvii (DSM 44981) were incubated in DSMZ medium 65  supplemented with 0.35% octocrylene at a temperature of 30 °C.Several M. agri and G. cholesterolivorans was modificated in order to enhance the biofilm formation. Therefore, the carbon sources of the medium 645 of M. agri were highly decreased to 0.05% glucose and 0.05% glycerol and BSA was also reduced to 0.05%. The carbon sources of medium 65 of G. cholesterolivorans were decreased to 1 g/L glucose and 1 g/L yeast extract . Incubation was performed in the presence of 0.35% octocrylene.The medium of the positive octocrylene-colonizing strains 9Reference standard octocrylene was purchased from Sigma Aldrich Ltd, Saint Louis, MO, USA (≥98%). Reference substances 2-(Carboxymethyl)butyl-2-cyano-3,3-diphenylacrylat (DOCCA), 2-Cyano-3,3-diphenylacrylic acid (CPAA) and 2-Ethyl-5-hydroxyhexyl-2-cyano-3,3-diphenylacrylate (5OH-OC) were provided by V.N. Belov, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany. Preparation and purity (>95%) are well described in ref. Hexane and dichloromethane were obtained from Merck KGaA, Darmstadt, Germany. Acetonitrile (LC-MS grade) was obtained from Merck, Darmstadt, Germany, formic acid (additive for LC-MS) was purchased from Honeywell Fluka, Muskegon, USA.All reagents were of analytical grade. Deionized water was purified using a Milli-Q-system .M. agri and G. cholesterolivorans (and controls) (20 mL) was transferred to a centrifuge tube and 10 mL hexane/dichloromethane (1 + 2) was added. After 30 min stirring the sample was centrifuged (6000 rpm). The lower layer was stirred with NaSO4 and centrifuged  again. For GC-MS analysis the liquid was filled in a microbial. For LC-MSMS analysis 1 mL was filled in a volumetric flask, evaporated and refilled with acetonitrile.The whole incubation culture of Quantification of octocrylene and qualitative recording of products: The quantitative determination of octocrylene and qualitative recording of the products were carried out on an Agilent Technologies GCMS system 5977 A MSD with Gerstel KAS4 injection system. The mass selective detector (MSD) was operated in EI mode. All system operations were controlled by MassHunter B.07.001413. Separation and quantification were performed on an Agilent DB5MS ultra inert capillary column, 30 m × 0.25 mm × 0.25 µm film thickness. Sample volumes of 1 µL were injected in split mode (1:5) at 1 mL/min flow. GC system inlet and MS interface temperatures were set at 70 and 280 °C, respectively. The column temperature was programmed as follows: held at 70 °C for 2 min, then ramped up to 140 °C at 10 °C/min, held for 3 min, then to 300 °C at 100 °C/min. The total runtime was 17,6 min. The carrier gas was helium with constant flow of 1 mL/min, the MSD operating parameters were routinely set by tune file. EM potential was set at +1352 V, MS source temperature at 230 °C and the quadrupoles at 150 °C.m/z 249 and qualifying fragment ions m/z 360 and 204. Recording of products was done by TIC mode .Quantitative determination of octocrylene was done by SIM mode, with quantifying ion with Stock solution was prepared with a concentration of 5300 mg/L using dichloromethane as solvent. By diluting the stock solution with hexane-dichloromethane (1 + 2) calibration standards were prepared with levels from 5,0 mg/L to 200 mg/L. While the stock solution was stored at −18 °C, the calibration standards had to be newly made for each analytical run.Instrumentation and parameters: The quantitative determination of metabolites was carried out on an Agilent 1260 HPLC system , equipped with binary pump, degasser, thermostat autosampler, column oven, which is coupled to the Sciex Qtrap 5500 tandem mass spectrometer .Separation was achieved by using a Phenomenex Synergi Fusion RP 80 A column  with a Phenomenex standard C18 guard column. The mobile phase consisted of Milli-Q water with 0.1% formic acid (A) and acetonitrile with 0,1% formic acid (B), the flow rate was set to 0.7 mL/min. The gradient elution was programmed as follows: 0–3.5 min, 40% to 95% B (acetonitrile), 3.5–7.0 min 95% B, 7.1–10 min, 40% B.The injection volume was 5 µL, the column temperature was set to 35 °C.To detect all three available metabolites with at least two characteristic mass transitions per analyte in one run, the electrospray source of the tandem mass spectrometer operated simultaneously in positive and negative ion mode Table .Table 1CMass transitions, declustering potentials and collision energies for the compounds were obtained by direct infusion experiments with single compound standard solutions.The following mass spectrometer settings were used: Curtain gas 40 psi, source temperature 450 °C, ion spray 4500 V, −4500 V respectively, Gas 1/Gas2: 40/60 psi.Stock solutions were prepared with a concentration of 100 mg/L using acetonitrile. By diluting a mix standard solution (concentration 10 mg/L) calibration standards were prepared with acetonitrile/water (60/40) at levels from 2,0 µg/L to 100 µg/L. While the stock solutions were stored at −18 °C, the calibration standards had to be newly made for each analytical run.9.Due to a substantial peak separation, the identity of peaks could be confirmed using retention times and mass transitionsData were acquired and processed on Analyst 1.6.3. Quantification was carried out with an external calibration. The calibration curves were calculated by linear regression with a RSD of <20% and a coefficient of determination (R2) of >0.99.20.All biodegradation assays were performed in biological triplicates. The dot-plot format of Fig. Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


To explore the value of diffusion kurtosis imaging (DKI) and histogram analysis for assessing preoperative stages and heterogeneity in rectal cancer.  Fifty patients with pathologically confirmed rectal adenocarcinoma were enrolled. The value of DKI parameters and histogram metrics for assessing the preoperative stages and heterogeneity in rectal cancer was analyzed retrospectively. −3 mm2/s versus 0.58 ± 0.11 × 10−3 mm2/s and 0.73 ± 0.11 × 10−3 mm2/s versus 0.65 ± 0.11 × 10−3 mm2/s; p values were 0.035 and 0.024, resp.). (2) D-10th percentile and D-25th percentile were also significantly higher in T1-2 than in T3-4 rectal cancer . (3) K value and its histogram metrics showed no statistically significant difference between T1-2 and T3-4. (4) D-10th had the largest area under the curve (AUC 0.799) among all the parameters; the sensitivity and specificity were 84.2 and 61.3%, respectively. (5) DKI combined with traditional MRI had an accuracy of 68% while assessing the lymph node of rectal cancer.  (1) ADC-10th percentile and ADC-25th percentile were significantly higher in T1-2 than in the T3-4 rectal cancer (the ADC values were 0.65 ± 0.08 × 10D-10th percentile exhibits the highest diagnostic efficiency. DKI parameters and histogram metrics are rather valuable in assessing the preoperative stages of rectal cancer;  Rectal cancer is one of the most common malignant tumors in the gastrointestinal tract, which accounts for approximately 50%–70% of colorectal cancers. With the advances in the society, economic status, and changes in people's diet, the incidence of rectal carcinoma has increased gradually in the world , 2. Thusb values, DKI sequences, and conventional T1WI/T2WI sequences. The radical surgical resection and pathology staging results were clear  [From March 2016 to April 2017, the patients with pathologically confirmed adenocarcinoma were selected at our hospital. The inclusion criteria for the patients were as follows. MRI examinations were fulfilled including multiple edition) ; (c) onlb values of 200, 500, 1000, 1500, and 2000 s/mm2). The detailed scan parameters were as follows: oblique axis T2WI: TR (time of repetition) = 3770 ms, TE (time of echo) = 101 ms, PAT  = 2, FOV (field of view) = 200 × 200 mm2, matrix size = 128 × 128, slice thickness = 4 mm, and number of sections = 20; oblique axis T1WI: TR = 700 ms, TE = 12 ms, PAT = 2, FOV = 280 × 280 mm, matrix size = 128 × 128, slice thickness = 4 mm, and number of sections = 20; and DKI: TR = 4900 ms, TE = 87 ms, flip angle = 90°, PAT = 4, FOV = 280 × 280 mm2, matrix size = 128 × 128, voxel size = 1.1 × 1.1 × 5 mm, slice thickness = 4 mm, number of sections = 20, and acquisition time = 260 s. In addition, the largest b value of DKI was 2000 s/mm2 [The MRI examinations were performed using a 3.0 T MR scanner  with a dedicated 32-channel pelvic phased-array coil to optimize signal-to-noise ratio. Before MRI, all the patients underwent bowel cleaning. A dose of 20 mg of the spasmolytic agent hyoscine butylbromide  was administered intravenously to all patients immediately to minimize bowel peristalsis and avoid motion artifacts. The MR sequences consisted of the oblique axis  T1-weighted imaging, oblique axis T2-weighted imaging, and oblique axis DKI sequences . The pixel-wise ADC value, equation , 11:(1)equation , 12:(2)ian bias , 15, incp values < 0.05 were considered as statistically significant. All parameters were first tested by the Kolmogorov-Smirnov test for normality analysis and the Levene's test for variance homogeneity. Independent sample t-test was used to analyze and compare the parameters between pT1-2 and pT3-4. The receiver operating curve (ROC) was used to assess the diagnostic performance of the parameters, and the area under the receiver operating characteristic curve (AUC), sensitivity, and specificity was calculated. The cut-off value was calculated by maximizing the Youden's index. The intraclass correlation coefficient (ICC) was used to evaluate the stability of the parameters between the two observers.The mean values of all the parameters measured by the two radiologists were used in the statistical analysis conducted using the statistical software SPSS 23.0 . Thirty-five patients underwent a low anterior resection, twelve patients underwent an abdominoperineal resection, and three patients underwent an extended resection. Among the 50 patients with tumors, 19 tumors were T1-2 (38%) and 31 were T3-4 (62%), according to the results of the surgical specimen analysis. Moreover, 53 lymph nodes were positive, according to the pathological results. MRI  showed 36 positive lymph nodes. The accuracy of DKI coupled with conventional sequences in identifying positive lymph nodes was 68%. The results are summarized in −3 mm2/s versus 0.58 ± 0.11 × 10−3 mm2/s and 0.73 ± 0.11 × 10−3 mm2/s versus 0.65 ± 0.11 × 10−3 mm2/s; p values were 0.035 and 0.024, resp.). In addition, among all the indices of D value, D-10th percentile and D-25th percentile were significantly higher in T1-2 than in T3-4 rectal cancer . No statistical difference was observed in all the kurtosis  indices between early and late rectal cancer , whose cut-off value, sensitivity, and specificity were 0.875, 84.2%, and 61.3%, respectively. The D-10th percentile value showed an 84.2% sensitivity. According to the results, the 25th percentile ADC value showed the highest specificity of 80.6%. The results were summarized in Among the DKI histogram indices, ADC-10th, ADC-25th, D value, K value, and its histogram indices were consistent among the two interpreters. The interobserver agreement (ICC) of some histogram indices was shown in The ADC value, D and ADC values and higher K value. In our results, the D and ADC values were statistically higher in early rectal cancer than in the advanced group, and the K value was lower in both groups, corresponding to the theory of water molecule diffusion in the tissue as described above. Some previous studies reported that the ADC value was much higher in advanced rectal cancer than in the early stage [D-10th were 0.799, 84.2%, and 61.3%, respectively, in differentiating early and advanced rectal cancer. As to ADC-10th, the AUC, sensitivity, and specificity were 0.699, 78.9%, and 51.6%, respectively. Thus, the diagnostic performance of D-10th is superior to the ADC-10th in assessing T stages of rectal cancer based on the theoretical characteristics of DKI. So, DKI can more accurately reflect the diffusion of water molecules of biological tissues. Although no statistical differences in K value were observed between T1-2 and T3-4, it showed the trend to the higher K value with the higher T stage of rectal cancer. And similar result had been reported [DKI was first proposed by Jensen et al.  to accouly stage , 17. Howly stage , 10, 19 D-25th percentile might represent the area of lower D value in a part of the heterogeneous tumor mass [In this study, we used whole-tumor volume histogram analysis, which analyzed the smallest unit of a voxel in every section that contained lesions. The current results demonstrated that both the 10th and 25th percentile values of ADC and diffusivity were lower in T3-4 than in T1-2 rectal cancer, albeit the differences were not statistically significant. In advanced rectal cancer, the necrotic cystic area was primarily in the center of the tumor. In the necrotic cystic area, the membrane tumor cell may lose its integrity. Consequently, the diffusion of water molecules was not restricted, indicating the high value of ADC and diffusivity. Furthermore, on the edge of the tumor, the tumor cell hyperplasia is rather active, and the tumor cell atypia is distinct; the tumor tissue microstructure is complex, and the water molecule diffusion is complex and restricted. Therefore, the values of ADC and diffusivity are lower on the edge of tumor mass that might represent the 10th and 25th percentile values according to the histogram analysis. A similar study also demonstrated that the mor mass . In the b value DKI sequences exhibited a superior potential, although more large sample studies are imperative to substantiate the value of DKI.Inside the involved lymph node of rectal cancer patients, the normal tissue is replaced by tumor cells, followed by an increase in the cell density. Therefore, the microstructure is complex, and the diffusion of water molecule is restricted, thereby indicating that the lymph node is distinctly shown as a high signal in the DKI sequence. The precise staging of lymph nodes is critical to the choice of clinical treatment, thereby necessitating the improvement of the accuracy of lymph node staging. Among the current methods of examination, MRI has a greater advantage in lymph node staging. MRI has the advantage of multiple sequences and parameters, including conventional sequences, diffusion-weighted imaging (DWI), and the dynamic contrast-enhanced sequence. However, the accuracy of the method failed to satisfy the clinical need. The study by Doyon et al.  showed tD-10th percentile was mostly accurate in distinguishing T1-2 from T3-4, which indicates that D value may serve as a potential biological indicator to evaluate the preoperative staging of rectal cancer noninvasively. Also, the study has some limitations. Firstly, this was a retrospective study, and hence, a selection bias would be inevitable. Secondly, the sample size was relatively small. Therefore, a large sample and multicenter study are essential to further verify the value of DKI.In worldwide, DKI is an interesting technique especially in breast cancer, prostate cancer, and cervical cancer. As in rectal cancer, DKI focused on histological grade and the evaluation of neoadjuvant chemoradiation in which DKI shows good performance and the potential application value in rectal cancers. In our study, the major finding of this study is that we used whole-volume histogram analysis to evaluate the value of DKI parameters in preoperative staging and heterogeneity of rectal cancer. The results showed that


The vortex fluidic device (VFD) is a thin film microfluidic platform which has a wide range of applications in synthesis and other areas of science, and it is important to understand the nature of the thin film of liquid in its inclined rapidly rotating tube. Neutron imaging has been used to determine the thickness of the film in a quartz tube with its shape modelled mathematically, showing good agreement between the model and experiments. The resultant equations are useful for studying VFD mediated processing in general, for which the optimal tilt angle of the tube is typically 45°. This includes its utility for the intelligent scale-up of organic syntheses, as demonstrated in the present study by the scaling up of an imine and amide synthesis to >1 g/min. It is able to refold proteins1, accelerate enzymatic and chemical reactions4, fabricate different nanocarbon materials6, incorporate drugs into bilayers, and a growing number of other applications. Despite these capabilities, optimisation of these processes is challenging without an understanding of the fluid behaviour within the VFD, particularly when attempting to scale the processes up to larger scales. This thin-film microfluidic platform features a hollow open-ended cylindrical tube  rapidly rotated at speeds, typically up to 9000 rpm. Liquid placed within the tube forms a thin film against the glass as a vortex to the bottom of the tube in an approximately a paraboloidal shape, and experiencing shear stresses on the order of 1 Pa1. The processing environment can be controlled by altering the tilt angle of the axis of the tube in relation to the horizontal (θ) position, the rotational speed (ω), and other operating parameters of the device  Fig.  is a rem7. Under continuous flow (CF), liquid is continuously delivered to the bottom of the tube through feed jets inserted at the top, or at strategic positions along it for multi-processing in a single VFD7. Once liquid in the film reaches the top of the tube, it exits the tube and is collected through a Teflon housing unit8. In general, for flow chemistry, the residence time is an important factor in controlling chemical and enzymatic reactions, and in controlling the formation of various carbon materials10. This often has a dramatic effect on the outcome of the processing, including in the VFD8. In most continuous flow systems, calculation of the residence time is straight-forward because the volume of the system is only affected by the number and size of the continuous flow processors. In contrast, the volume within the VFD is also affected by the rotational speed and tilt angle, causing the volume calculation to be more complicated. In order to calculate the residence time and better understand and design the processing, it is necessary to understand the shape of the film and how this is influenced by rotational speed, tilt angle, as well as the volume of liquid in the tube. The derivation of these equations allows quantitative answers to questions regarding the maximum volume the tube can hold in confined mode , as well as the selection of operating conditions to give the desired residence time under continuous flow, allowing the intelligently-designed scale-up of chemical reactions.For the confined mode (CM) of operation of the VFD, a specific volume of fluid is placed within the tube, which is then capped and processed for a specified time12, as well as other work performed investigating the film shape13. The model is further detailed in the Supplementary Information, with the resultant equation for the film heightr is the radial distance from the rotation axis of the tube and z is the distance along this axis above the base of the tube. The location of the film is influenced by the tube radius (R0), the rotational speed (ω rad/s), gravitational acceleration (g), the tilt angle (θ), and the volume of the fluid (V). The equations presented here are asymptotic approximations valid for high rotation rates (ω2R0g) that, in the range of interest, agree with the full equations (presented in the SI) to better than 95%.The model takes into account gravity and the centripetal force, and is consistent with previous derivations for Liquid Mirror Telescopes, which use the reflective paraboloid produced by mercury in a rotating container as an astronomical telescopeR0 = 8.75 mm , θ = 45° and ω = 6000 rpm (628 rad/s), the film is thin with ftR0 and the thickness decreases almost linearly with height, Fig. R0 = 4.75 mm. In addition to determining the film thickness, it is important to know the maximum film height hm because when this equals the height of the tube H, the volume of liquid in the VFD for CF is reached. When this volume is exceeded under CM, the liquid contacts the cap.Under typical operating conditions of the VFD, with z starts at zero, when the rotation rate is still too low for the film to have reached this height. Then as the rotational speed increases, the film reaches this height producing a thin film. As the rotational speed is further increased, a thicker portion of the film is present at a given z, however the decrease in average film thickness partially counters and eventually overcomes this increase, leading to a decrease in the film thickness. Under particular operating parameters, it is important to maximise the film thickness at a particular height z, and thereby the rotational speed ωm affording the greatest film thickness is given byωm (7575 rpm) giving the maximum film thickness at the laser height (z = 8.5 cm).For applications of the VFD in chemical processing and in other areas of science, it is important to determine the conditions that result in the maximum film thickness for specific points along the tube. In considering the effect of rotational speed on film thickness for a particular height for a given volume of fluid, as depicted in Fig. Vm the device can contain under CM without it interacting with the cap at the top of the tube; this volume is also the volume within the device under CF mode of operation, and is given byτ under CF) is proportional to the inverse of the square of the rotational speed. This can be easily converted into an equation for the residence time τ based on the flow rate Q, using the equation τ = VQ−1.An important result of the model is that there is a maximum volume ω = 6950 rpm (728 rad/s) at θ = 45°14. Re-analysing the data in the light of the present results indicates that a fit of τ = 158.57Q−1 results in a stronger correlation (as determined by the R2 correlation coefficient) than the originally reported fit of τ = 152.37Q−0.934 . In comparing this experimental result to theoretical calculations, it is important to note that the experimentally determined residence time includes time spent within the Teflon collection unit at the top of the tube and the outlet pipette used to collect the liquid, which is not considered in the model. This gives the residence time asVE is the volume within the system that is external to the tube. Equating this to the experimentally determined formula of τ = 158.57Q−1 results in a VE value of 1.16 mL, which is a reasonable estimate of the volume of the liquid residing in the Teflon unit and pipette. For a flow rate of 1 mL/min, the difference between this model and the previous correlation is less than 5%, but this relative difference is higher for lower flow rates (9% for 0.5 mL/min and 21% for 0.1 mL/min).Previous work involving the optimisation of biodiesel synthesis using the VFD required experimentally measuring the residence time as a function of flow rate for 2. Additionally, it is revealed that for multi-step processing within a single tube, the residence time τp for a section from z = z1 to z = z2 is15.The developed model also provides insights into better understanding earlier processes performed within the VFD. Previously, the effect of rotational speed on the volume within the tube under continuous flow has been assumed to be minimal, in contrast to the model presented here. However, because increasing the rotational speed significantly decreases the volume within the tube, it is expected that a general decrease in yield may be observed with an increase in rotational speed for continuous flow processes due to the decreased residence time. This effect has been observed in previous esterification procedures, where it was instead attributed to evaporation of the solventin situ. Spectroscopic approaches were unsuccessful because the tapered nature of the thin film along the tube resulted in refraction, eliminating its potential for determining the film thickness. However, the unique imaging capabilities of neutrons enabled the measurement of both the film thickness and maximum height of the film extending up the tube.The model was tested in developing an analytical technique to determine the film thickness 16. Consequently, air and water can be visually distinguished, despite having similar spectroscopic properties. Additionally, some metals  are essentially transparent to neutrons, allowing easy subtraction of the VFD itself from the image.Neutron imaging is a radiographic testing technique for viewing the interior of objects where the degree to which different elements attenuate (scatter or absorb) the probing neutron beam allows the visualisation of systems that are otherwise difficult or impossible to view by other methods. This is due to the particular physical properties of neutrons in comparison to other elementary particle probes. Neutrons are uncharged; they can deeply penetrate into matter and interact with the nucleus of an atom rather than with the diffuse electron cloud. Therefore, in neutron imaging techniques, the contrast is based on the ability of an element to attenuate neutrons, which is not linearly dependent on atomic number2 = 0.69), despite significant scatter due to the limited resolution for the film thickness measurements. In contrast, the maximum film height  with the values predicted from.3.The thin film produced in the VFD for 1 mL of water under confined mode at various rotational speeds was visualised under neutron imaging , with the same 20 mm O.D., and by minimising the rotational speed used. The minimum rotational speed capable of maintaining a vortex to the base of the glass tube is such that ft = R0 at z = 0, giving Previous continuous flow operation of the VFD has involved a maximum flow rate of 2 mL/min for fast and often exothermic reactions, whereas typical flow rates are approximately 0.5 mL/min15, which corresponds to a flow rate of 10 mL/min under the scaled-up design. The synthesis of three imines under this setup produced conversions of 82%, 87%, and 90%  will have reduced micromixing and shear stress, resulting in a less efficient reaction. Nevertheless, the yields remained higher in two of the three cases.Similarly, the previous synthesis of amides used a residence time of 40 secondsIn conclusion, we have developed a mathematical model describing the shape of the film in the VFD that is consistent with the experimentally determined film thickness as determined using neutron imaging. However, this novel approach has limitations in measuring film thickness at high rotational speeds of the VFD. Applying the model to previous work has elucidated the fact that under continuous flow, the increased residence time at low rotational speeds may result in higher yields than in the presence of pressure waves at high speeds. It was also determined that translating optimum conditions for the confined mode of operation of the device to continuous flow, a strategy which has served us well in some earlier research, may be an over simplification. The mathematical model was also used to design the scaled-up conditions for the synthesis of imines and amides, affording production rates ranging from 22 g/hour to 92 g/hour, which is a significant increase from previous procedures.6LiF scintillation screens. The field of view was adjusted to a nominal pixel size ≈ 67 μm by using a Carl Zeiss lens with a fixed focal length of 50 mm18. Exposure time for the camera was 1 s, with an additional 0.02 s of read out time required to transmit the data, resulting in a sampling frequency of 0.98 Hz. Initial investigations indicated that liquid within the VFD reached an equilibrium point after 5 seconds. The device was set to alternate between operation for 10 s and halt for 10 s, with each operation period increasing in rotational speed by 50 rpm. The device was operated at speeds from 2000 rpm to 4800 rpm. The raw images were processed using the program ImageJ, and 3 images were used to produce the normalised images; background signal DF , blank OB , and sample I . Each image was preliminarily treated by applying a despeckle noise filter, and the normalised image In was produced by subtracting the background from both the blank and sample images, followed by dividing the sample image by the blank:Experiments were performed on the DINGO neutron beam line at the Australian Nuclear Science and Technology Organisation (ANSTO) OPAL research reactor at Lucas Heights, NSW, Australia. The instrument features a thermal neutron beam gathered through in-pile collimators positioned upstream of the beam line. In the case of real-time imaging of dynamic process, the inlet aperture of 20 mm in diameter (D) is configured to provide high flux and high spatial resolution (L/D of 500) at the length path (L) from the collimator to the sample position. The detector system was equipped with the CMOS camera  × 2160(h) pixels sensor) coupled with 100 μm thick ZnS/1 and 2 formed an oil, and the water was split into 250 mL fractions, each washed with ethyl acetate (200 mL x3), recombined, dried (MgSO4), and the solvent removed under reduced pressure and in vacuo to give 1 as a yellow oil  and 2 as a solid. Compound 2 was subsequently vacuum filtered to give an impure yellow solid  which was recrystallised from ethyl acetate affording an off white solid . Compound 3 formed a solid, which was filtered, dissolved in ethyl acetate, dried (MgSO4), and the solvent removed under reduced pressure. Cooling gave an impure yellow solid  which was recrystallised form ethanol affording pale yellow crystals .55 mL solutions of amine and aldehyde were prepared in methanol at 1.94 M. The 38 cm long VFD was operated at 2970 rpm  and each solution was injected into the VFD at 5 mL/min for 10 minutes. The VFD was subsequently operated for an additional 3 minutes to allow all injected reagents to experience 3 minutes within the device. Output from the VFD was quenched in 500 mL of gently stirring water. Imines 4), and solvent removed under reduced pressure, giving 4 as a solid and 5 and 6 as liquids. Amide 4 was vacuum filtered and recrystallised from ethanol affording a white solid . Amides 5 and 6 were dried in vacuo and recrystallised from ethanol affording 5 as a white solid , and 6 as a transparent solid .55 mL solutions of amine (0.43 M) in chloroform with 7% v/v triethylamine were prepared, along with 55 mL solutions of acylchloride (0.755 M) in chloroform. The VFD housing a 38 cm long glass tube was operated at 4500 rpm and each solution was injected into the VFD at 10 mL/min for 5 minutes. The VFD was subsequently operated for an additional 40 seconds to allow all injected reagents to experience 40 seconds within the device. Output from the VFD was quenched in 500 mL of gently stirring 2 M HCl. The organic layer was separated from the aqueous layer, dried : δ 8.42 , 7.82 , 7.45–7.44 , 7.37  = 4.4 Hz, 4 H), 7.31–7.28  = 4.2 Hz, 1 H), 4.86 . 13C NMR : δ 162.1, 139.3, 136.2, 130.8, 128.7, 128.5, 128.3, 128.0, 127.0, 65.1. This is consistent with literature14.1H NMR : δ 8.99 , 8.86  = 9 Hz, 1 H), 7.89–7.88 , 7.59–7.56 , 7.51–7.49 , 3.31  = 10.4 Hz, 4.1 Hz, 1 H), 1.87 , 1.71 , 1.44  = 12.4, 3.3, 2 H), 1.33  = 12.3, 3.5, 1 H). 13C NMR : δ 157.9, 133.8, 132.2, 131.4, 130.6, 128.6, 128.2, 126.9, 125.9, 125.3, 124.3, 70.9, 34.6, 25.8, 24.8. This is consistent with literature14.1H NMR : δ 8.47 , 7.92–7.90 , 7.49–7.47 , 7.40  = 7.8 Hz, 2 H), 7.25–7.21 . 13C NMR : δ 160.4, 152.1, 136.2, 131.4, 129.2, 128.83, 128.79, 126.0, 120.9. This is consistent with literature14.1H NMR : δ 7.51  = 7.9 Hz, 2 H), 7.31  = 7.4 Hz, 2 H), 7.17 , 7.10  = 7.4 Hz, 1 H), 2.34  = 7.5 Hz, 2 H), 1.77  = 7.4, 2 H), 1.01  = 7.4 Hz, 3 H). 13C NMR : δ 171.2, 138.0, 129.0, 124.2, 119.8, 39.8, 19.1, 13.8. This is consistent with literature19.1H NMR : δ 7.34  = 7.4 Hz, 2 H), 7.28–7.27 , 5.67 , 4.45  = 5.7 Hz, 2 H), 2.20  = 7.5 Hz, 2 H), 1.70,  = 7.4 Hz, 2 H), 0.96  = 7.4 Hz, 3 H). 13C NMR : δ 172.8, 138.4, 128.7, 127.9, 127.5, 43.6, 38.8, 19.2, 13.8. This is consistent with literature17.1H NMR : δ 5.26 , 3.77 , 2.11  = 7.5 Hz, 2 H), 1.92–1.90 , 1.72–1.68 , 1.65  = 7.5 Hz, 2 H), 1.63–1.59 , 1.36  = 12.5, 3.4 Hz, 2 H), 1.18–1.07 , 0.94  = 7.4 Hz, 3 H). 13C NMR : δ 172.0, 48.0, 39.1, 33.3, 25.6, 24.9, 19.3, 13.7. This is consistent with literature20.Supplementary Information