And three of them, Impurity-2, Impurity-3, and Impurity-5, have never been reported before. The fragmentation patterns of five impurities were proposed on a basis of accurate mass and fragment ions in this study. Since the toxicity of impurities is relevant to their structures, toxicology of all five impurities was predicted by three software tools, and the result showed that these compounds have good safety profile.Pharmaceutical companies and regulatory agencies have more and more concerns for impurities in pharmaceuticals and their toxicity. In this work, heart-cutting two-dimensional ultrahigh-performance liquid chromatography (2D-UHPLC) in combination with high-resolution mass spectrometry (HRMS) was used, setting HRMS as positive mode of electrospray ionization to identify five impurities in pioglitazone hydrochloride preparations. With the heart-cutting 2D-UHPLC and online desalting technique, the structures of five impurities were deduced in an analysis of MS The cleavage product at m/z 240 is a loss of 2,4-thiazolodinedione (-C3H3NO2S) from [M + H]+, and m/z 286 can be attributed to the ring opening of 2,4-thiazolodinedione to remove oximide (-C2HNO2). The product ions at m/z 134, 119 were the characteristic product ions derived from the cleavage of ethoxy phenyl ether bond.Fragmentation pattern of pioglitazone is beneficial to elucidate the structures of target impurities. HRMS analysis of pioglitazone showed a protonated molecular ion peak at m/z 258.1480 [M + H]+ observed in the HRMS spectrum of Impurity-1 was matched to the molecular formula C16H19NO2 (exact mass: 257.1410). 2 spectrum of m/z 258.1480. Characteristic product ions of Impurity-1 at m/z 134 and 240 indicated that its structure is unchanged compared with pioglitazone except 2,4-thiazolodinedione unit. The characteristic product ions at m/z 240 and 228 were corresponding to the loss of H2O, CH2=O from [M + H]+, respectively, indicating the presence of hydroxymethyl group (-CH2OH) in the structure of Impurity-1. The structure of Impurity-1 is shown in + for Impurity-1.A protonated molecular ion peak at Impurity-1 is a process impurity. As shown in m/z 272.1272 [M + H]+ corresponding to the molecular formula C16H17NO3 (exact mass: 271.1203). 2 spectrum of m/z 272.1272. Characteristic product ions of Impurity-2 at m/z 134, 119, and 228 indicated that its structure is unchanged compared with pioglitazone except 5-methylene-2,4-thiazolodinedione unit. The characteristic product ions at m/z 254 and 228 were the loss of 18 Da and 44 Da from protonated molecular ion of Impurity-2, respectively, indicating that the structure of Impurity-2 contains carboxyl group (-COOH). The structure of Impurity-2 is shown in + for Impurity-2.The HRMS spectrum of Impurity-2 showed a protonated molecular ion peak at Intermediate-1 may be oxidized into Impurity-2 under alkaline conditions. Thus, Impurity-2 is a process impurity.m/z 373.1205 [M + H]+ which is compatible to the molecular formula C19H20N2O4S (exact mass: 372.1138) which has the same molecular formula of impurity A  group. Therefore, substituted position of the hydroxyl group was altered comparing with impurity A. The structure of Impurity-3 is shown in + for Impurity-3.The HRMS analysis of Impurity-3 displayed a protonated molecular ion peak at purity A  listed ipurity A .  FigureAccording to the mechanism of Knoevenagel condensation reaction, Impurity-3 was the by-product of the synthetic process of Intermediate-2 . Therefom/z 341.1311 [M + H]+ obtained in the HRMS spectrum of Impurity-4 was consistent with the molecular formula C19H19N2O2S (exact mass: 340.1240). 2 spectrum of m/z 341.1311. The exact mass of Impurity-4 was 18 less than pioglitazone, which denoted the loss of H2O. Furthermore, the distinction between Impurity-4 and pioglitazone was the 2,4-thiazolodinedione unit owing to the presence of typical fragment ions at m/z 134 and 240. Therefore, a loss of H2O occurred on the 2,4-thiazolodinedione. The presence of a fragment ion at m/z 114 manifested that the thiazole ring became more stable corresponding to the increased stability by the formation of carbon-carbon double bond after the dehydration of the C-4 position carbonyl. The structure of Impurity-4 is shown in + for Impurity-4.A protonated molecular ion peak at During the process of Intermediate-2 being reduced to pioglitazone, it may be over-reduced and then dehydrated to form Impurity-4 . Therefom/z 357.1259 [M+H]+ corresponding to the molecular formula C19H20N2O3S (exact mass: 356.1189), and this showed that Impurity-5 is the isomer of pioglitazone. 2 spectrum of m/z 341.1311. The presence of fragment ions at m/z 134 and 240 indicated that its structure is unchanged compared to pioglitazone except 5-methylene-2,4-thiazolodinedione unit. The possible structure of Impurity-5 is deduced (+ for Impurity-5.The HRMS data of Impurity-5 showed a protonated molecular ion peak at  deduced  by referImpurity-5 is a process impurity. The 4-position carbonyl group of the 2,4-thiazolodinedione ring might be reduced in the process of Intermediate-2 reduction .As exhibited in The value of TOX_Risk predicted by ADMET Predictor™ indicated the number of potential toxicity problems that a compound might have. The predicted compounds with the value below 3.3 are considered as safe. The online desalting technique achieved by heart-cutting 2D-LC coupled with HRMS showed several advantages in structural identification of impurities. First, the aimed impurity can be transported into mass spectrometry without changing the mobile phase of the analytical method which may contain nonvolatile salt or high concentration salt. In addition, HRMS can offer information of accurate mass and secondary fragment ions which are helpful for structural elucidation. In this study, fragmentation patterns of pioglitazone and five unidentified impurities were investigated and applied to obtain structural information of these impurities. Two impurities, Impurity-1 and Impurity-4, were reported previously, whereas the remaining three were first reported in this article. The toxicity assessments of these five impurities were predicted, which indicated that they all have a good safety profile. This study may provide a reference for the quality control of pioglitazone hydrochloride preparations.


Background: Considering the threat of antimicrobial resistance (AMR), Ethiopia implemented strategies to combat AMR, including partnering with the American Society for Microbiology (ASM) to conduct an AMR training program using the Project ECHO learning platform. ECHO AMR was used to virtually connect subject-matter experts with participating sentinel laboratories in remote locations to provide ongoing education, telementoring, and foster peer-to-peer learning and problem-solving in microbiology. In phase 1, the ASM had primary leadership in conducting sessions and project administration. In phase 2, roles and responsibilities transitioned from the ASM to the Ethiopian Public Health Laboratory (EPHI) with support from ECHO India. Here we describe the transition process and lessons learned. Methods: From December 2020–2021, biweekly 1-hour sessions were conducted for 8 sentinel laboratories. Each virtual session included a lecture led by a subject-matter expert, a case presentation by a participating laboratory, open discussion, and feedback via an end-of-session online survey. Following a transition plan, initial ASM-EPHI transition activities included formal administrative and logistical training, including participation in a 3-day Project ECHO-immersion program provided by ECHO India. Selected administrative and technical roles and responsibilities, including further developing their own SMEs, were transitioned from ASM to EPHI every 4 sessions. ASM conducted postsession reviews with EPHI and ECHO India to discuss successes and suggested improvements. Results: Leadership of ECHO AMR was fully transitioned to EPHI over 12 months. End-of-session surveys and postsession reviews indicated the transition process was successful, with EPHI staff leading the lectures, session coordination, and facilitation, and positive feedback from session participants. Challenges included variable sentinel site participation due to competing priorities such as COVID-19 testing and poor internet connectivity during the rainy season. Lessons learned included the need to use a gradual transition strategy with close monitoring, training facilitators to maintain implementation fidelity (level of reproducibility to conduct ECHO AMR as in phase 1) and improve participation, and assessing individual learning, using pretests and posttests. Recommendations included that ASM should remain as an external technical advisor to ensure program technical depth and session facilitators be trained to improve participation in the discussions. Implementation fidelity compared to phase 1 was considered moderate, with the gap primarily due to the need for dedicated release time from laboratory duties to ensure session leadership, coordination, and facilitation. Conclusions: Leadership and laboratory workforce capacity-building responsibility for AMR training was successfully transitioned from ASM to EPHI, promoting self-sufficiency in training and with far-reaching benefits in the global fight against AMR.Disclosures: None


Background: Efficient theranostic strategies concurrently bring and use both the therapeutic and diagnostic features, serving as a cutting-edge tool to combat advanced cancers.Goals of the Investigation: Here, we develop stimuli-sensitive theranostics consisting of tailored copolymers forming micellar conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds, thus enabling the tumor microenvironment-sensitive activation of the photodynamic therapy (PDT) effect, fluorescence or phosphorescence.Results: The nanomedicines show superior anti-tumor PDT efficacy and huge tumor-imaging potential, while reducing their accumulation, and potentially side effects, in the liver and spleen. The developed theranostics exhibit clear selective tumor accumulation at high levels in the mouse sarcoma S180 tumor model with almost no PyF found in the healthy tissues after 48 h. Once in the tumor, illumination at λexc = 420 nm reaches the therapeutic effect due to the 1O2 generation. Indeed, an almost complete inhibition of tumor growth is observed up to 18 days after the treatment.Conclusion: The clear benefit of the specific PyF release and activation in the acidic tumor environment for the targeted delivery and tissue distribution dynamics was proved. Conjugates carrying pyropheophorbide-a (PyF) attached by pH-sensitive hydrazone bonds showed their excellent antitumor PDT effect and its applicability as advanced theranostics at very low dose of PyF. The principle of the EPR effect relies on the leaky vasculature of tumor tissues and their vascular functions: tumor vasculature is characterized by large fenestrations between the endothelial cells formed during angiogenesis and by their irregular alignment. Moreover, they exhibit a dysfunctional lymphatic drainage, leading to the preferential extravasation/permeation into the tumor vasculature endothelium and the retention of macromolecules in the malignant tissue rather than the healthy one 2O2-responsive polymer prodrug NPs with GSH-scavenger were designed and prevented the depletion of 1O2, which induced the cooperatively strong oxidative stress and enhanced cancer cell apoptosis The encapsulation of several PSs into macromolecular systems, such as polymer nanoparticles Similarly, the covalent attachment of PSs to biocompatible and water-soluble polymer carriers forming polymer conjugates, which enhance the aqueous solubility of PSs, was described recently N-(2-hydroxypropyl)methacrylamide copolymers (pHPMA) have been extensively studied as carriers of bioactive molecules 1O2 generation, making them promising theranostic candidates. A combination of the pHPMA conjugate of PyF with vascular mediators, as nitric oxide and carbon monoxide Water-soluble and synthetic Herein, we designed, synthesized, and characterized micelle-forming pHPMA-based conjugates with PyF 5-hydroxy-2-pentanone ester (dPyF) covalently attached by the pH-sensitive hydrolytically degradable hydrazone bond. The stimulus sensitivity of these potential theranostics was successfully proved. The highly improved tumor-targeted delivery of dPyF was observed by fluorescence imaging. The pH-responsiveness and controlled activation of dPyF fluorescence and singlet oxygen production enhanced the treatment outcome with very low doses of polymer theranostics.bisisobutyronitrile (AIBN), methacryloyl chloride, 6-amino hexanoic acid, tert-butoxycarbonyl hydrazide, N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), carbon disulfide, ethanethiol, sodium hydride , 5-hydroxy-2-pentanone, 4,6-trinitrobenzene-1-sulfonic acid (TNBSA), pentafluorophenol, 4-(dimethylamino)pyridine (DMAP), tert-butanol, N,N-dimethylacetamide (DMA), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxy, free radical (4-oxo-TEMP) were obtained from Merck (Czech Republic). Pyropheophorbide-a was purchased from Frontier Scientific® (USA), 2,2′-azobis (V-70) from Wako Chemicals (Germany), and N-(3-tert-butoxycarbonyl-aminopropyl)methacrylamide (APMA-Boc) from Polysciences, Inc., (USA). All of the other chemicals and solvents were of analytical grade. The solvents for the nuclear magnetic resonance (NMR) characterization of DMSO-d6 (99.80 atom% D) and CD3OD (99.80 atom% D) were obtained from VWR Chemicals (Belgium).Chemicals such as 1-amino-propan-2-ol, 2,2′-azoFigure 1). The purity of the dPyF was confirmed by HPLC and 1H NMR, and the molar absorption coefficient was determined by UV-Vis spectrometry. The synthesis of the pentafluorophenyl ester derivative of PyF to prepare polymer conjugates with PyF bound by stable amide bonds was presented in our previous study.6Carbodiimide chemistry was used to prepare a derivative of PyF (dPyF) via the esterification of the PyF carboxylic group with the hydroxy groups of 5‑hydroxy-2-pentanone (N‑(3‑dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)  were dissolved in DCM (4 mL). Then, 5-hydroxy-2-pentanone  and the catalyst 4-(dimethylamino)pyridine (DMAP) were added, and the reaction mixture was continuously stirred for 3 h. The reaction was monitored using TLC , and the product was purified by a silica gel column (60 Å) using a gradient of CHCl3/MeOH, from 100/1 to 1/1 v/v, elution) . 1H NMR : δ = 9.37, 9.28, and 8.57 ppm ; δ = 7.91 ppm ; δ = 6.24-6.12 ppm ; δ = 5.26-5.08 ppm - from PyF); δ = 4.48 ppm ; δ = 3.97-3.89 ppm - from aliphatic linker); δ = 3.13 ppm - from aliphatic linker); and δ = 0.26 and 1.81 ppm .The synthesis of dPyF was as follows: PyF  and N-(2-hydroxypropyl)methacrylamide (HPMA) and N-(tert-butoxycarbonyl)-N'-(6-methacrylamidohexanoyl)hydrazine (MA-AH-NHNH-Boc) were synthesized according to the literature 1H NMR) (Monomers  1H NMR) .P1 bearing hydrazide groups poly(HPMA-co-MA-AH-NHNH2) was prepared by the controlled radical reversible addition-fragmentation chain transfer (RAFT) copolymerization of HPMA and MA-AH-NHNH-Boc using S-2-cyano-2-propyl-S`-ethyl trithiocarbonate as a chain transfer agent (CTA) and the initiator 2,2ʼ-azobis (V-70). The molar ratio of monomer/CTA/V‑70 was 360/2/1, and the molar ratio of monomers HPMA/MA-AH-NHNH-Boc was 90/10. Trithiocarbonate end groups were removed via reaction with an excess of 2,2′-azobisisobutyronitrile (AIBN), and Boc groups were thermally removed in Q-H2O as previously described P1 is described in Figure S1).The polymer precursor P2 containing amine groups poly(HPMA-co-MA-APMA) was prepared analogously by using HPMA and N-(3-tert-butoxycarbonyl-aminopropyl)methacrylamide (APMA-Boc), as previously described Table 1.The polymer precursor P1 were reacted with the dPyF in DMA in the presence of acetic acid overnight in the dark, resulting in polymer conjugates with dPyF bound by hydrolytically cleavable hydrazone bonds (P‑hyd-dPyF) . The purity of the conjugate was assessed using 1H NMR, and TLC was performed in a mixture of CHCl3 and methanol . The synthesis of P-hyd-dPyF containing a 5-hydroxy-2-pentanone spacer was as follows: The polymer precursor P1  and the dPyF  were dissolved in 3 mL of dry DMA. Then, 46 µL of acetic acid was added to the reaction, and the reaction mixture was continuously stirred at room temperature overnight in the dark. The conjugate was isolated and purified by precipitation into a mixture of ethyl acetate/CHCl3  three times. The precipitate was filtered off, washed twice with a mixture of ethyl acetate/diethyl ether , and dried under vacuum .The hydrazide groups of polymer precursor P-amide-PyF) was prepared using the polymer precursor P2 as previously reported Table 2.The polymer conjugate with PyF bound by stable amide bonds  and a C18 reversed-phase Chromolith Performance RP‑18e column  was used. Water/acetonitrile/0.1% TFA with a gradient of 5%-95% (v/v) acetonitrile at a flow rate of 4 mL min-1 was used as the eluent.High-performance liquid chromatography (HPLC) was used to assess the purity of monomers, chain transfer agent, and dPyF, and to monitor the rate of dPyF release from the conjugate DH) of polymer precursors P1 and P2, and polymer conjugates P-hyd-dPyF and P-amide-PyF . In the case of conjugates, a fluorescence filter was used for the measurements . Data were evaluated using the DTS(Nano) software, and five independent measurements were performed using samples from 1 to 3 mg mL-1 in PBS at pH 7.4 (filter 0.22 µm PVDF).Dynamic light scattering (DLS) was used to measure the hydrodynamic diameters (Mn), the weight-average molecular weight (Mw), and the dispersity (Đ) of polymer precursors P1 and P2 were determined by size exclusion chromatography (SEC) using a HPLC LC40 system equipped with SPD-M40 photodiode array , PSS Gram columns in series , Optilab‑rEX differential refractometer and DAWN HELEOS II multi-angle light scattering (MALS) detectors, . The analysis was performed using DMF + LiBr (0.5 g L-1) as a mobile phase at a flow rate of 1 mL min-1. The data were analyzed using the ASTRA VI software, and the refractive index increment dn/dc (for PHPMA copolymers in DMF ~0.09 mL g-1) was applied for the calculation of Mn, Mw, and Ð.The number-average molecular weight ( P-hyd-dPyF was prepared. Measurements were performed in duplicate by using the sample at 1.1 mg mL-1 in PBS (pH 7.4) at a flow rate of 1.0 mL min-1 by using an FFF (system Eclipse 3+) equipped with UV, DLS (DAWN Helios 8+), and Optilab-rEX differential refractometer index (RI)  detectors at λ = 658 nm. The ASTRA VI software was used, and the refractive index increment dn/dc (for pHPMA copolymers: ~0.167 mL g-1) was applied for the calculation of Mn, Mw, and Ð of the precursor.Field flow fractionation (FFF) was applied to evaluate the stability of the micelles formed when the conjugateP1 and P2, respectively, were determined in the borate buffer by using the 2,4,6-trinitrobenzene-1-sulfonic acid (TNBSA) assay method described before ε(NHNH2) = 17,200 L mol-1 cm-1 (λmax = 500 nm) for hydrazides and ε(NH2) = 11,550 L mol-1 cm-1 (λmax = 420 nm) for amines. The amount (wt.%) of dPyF and PyF in the conjugates was assessed in methanol by using the following molar absorption coefficients: in the case of P‑hyd‑dPyF, ε = 69,100 L mol-1 cm-1 (λmax = 416 nm) and 32,200 L mol-1 cm-1 (λmax = 668 nm) determined for the dPyF; in the case of P‑amide‑PyF, ε = 105,700 L mol-1 cm-1 (λmax = 416 nm) and 45,000 L mol-1 cm-1 (λmax = 668 nm) determined for the PyF compound. The samples' absorbance spectra were measured at 0.1 mg mL-1 PyF equivalent in methanol and recorded from 200 to 800 nm by using Specord 205 ST . The absorption spectra of PyF, dPyF and polymer conjugates are shown in The molar content of hydrazide and amine groups statistically distributed along the polymer precursors 1H NMR spectra were measured using a Bruker AVANCE III 600 spectrometer operating at 600.2 MHz by using DMSO-d6 for monomers and polymer conjugates, or CDCl3 for dPyF. To compare the molar content of dPyF in the conjugates with the results obtained by UV-Vis, the molar content of PyF in the polymer conjugates was calculated using the integral intensity of signals at δ = 6.23 ppm  and δ = 6.42 ppm  from the PyF molecule, and δ = 3.67 ppm  or 4.71 ppm  of the HPMA monomer unit. The 1H NMR spectra of monomers and polymers are listed in The morphology and dimensions of P-hyd-dPyF were examined using transmission electron microscopy (TEM). A 10 mg/ml solution of P-hyd-dPyF was prepared in deionized water. Subsequently, 50 μl of the solution was combined with 50 μl of 0.01% phosphotungstic acid for TEM analysis, using a JEM-1400 plus microscope manufactured by JEOL in Tokyo, Japan. The TEM image of P-hyd-dPyF is shown in P-hyd-dPyF was monitored by incubating the sample in a 0.1M phosphate buffer solution with 0.05M NaCl at pH values of 5.0 and 7.4 at 37°C, followed by extraction in chloroform as previously reported  v/v) and determined using an HPLC analysis. The relative area of peaks (absorbance at λ = 416 nm) was used for the calculation, and all of the drug-release data were expressed as the amount of free dPyF relative to the total dPyF content in the conjugate. All of the experiments were carried out in triplicate.The amount of dPyF released from the polymer conjugate P-hyd-dPyF, and P-amide-PyF were recorded using a spectrofluorometer model FP-6600, Jasco Corp., Tokyo, Japan. P-hyd-dPyF was dissolved in PBS in the absence/presence of the indicated concentrations of Tween-20, sodium dodecyl sulfate (SDS), lecithin, and urea. For comparison between polymer conjugates P-hyd-dPyF and P-amide-PyF with pure PyF, the samples were dissolved in PBS (pH 7.4) or in ethanol, and P‑hyd-dPyF was incubated in PBS (pH 5.0) with SDS 0.1% (w/v) at 37°C for 4 h. The sample solutions were prepared at 0.005 mg mL-1 dPyF equivalent, excited at λmax = 420 nm (corresponding to PyF), and the emission from 600 to 800 nm was recorded.The fluorescence spectra of pure PyF, P-hyd-dPyF and P-amide-PyF polymers in PBS were titrated to pure PBS at 37°C in 20 consecutive injections using a MicroCal ITC200 isothermal titration calorimeter . Additionally, blank titrations of P1 and P2 polymer precursors were performed in a similar manner. The heat for each injection (µJ) was plotted against the concentration of the polymer in the cell after the corresponding injection  dissociation to the overall heat of dilution, the heat from blank titration was subtracted from the heat of dilution of the polymer-porphyrin conjugate. Finally, the pH of all initial solutions was measured to account for the potential contribution of (de)protonation originating from the pH mismatch to the overall heat of dilution.The solutions of njection .5. The c1O2 generation was measured using electron spin resonance (ESR) spectroscopy. Solutions of P-hyd-dPyF were prepared in the concentration of 40 μg mL-1 PyF equivalent in the following solvents: (a) PBS at pH 6.5, (b) PBS at pH 7.4, (c) PBS at pH 5.0, (d) solution of Tween-20 at 0.5% (v/v) in PBS pH 7.4 and (e) after incubation in PBS (pH 5.0) with Tween-20 at 0.5% (v/v) at 37°C for 4 h. For all of them, 5% (v/v) of ethanol was added for dissolution. Then, 100 μL of 200 mM 4-oxo-TEMP (spin trapping agent) was added to 900 μL of the sample , which was placed into flat quartz cells . The illumination was carried out at different times (from 0 to 600 s) by using a xenon light source  from 400-700 nm. The X-band ESR spectra were recorded using a JEOL JES FA-100 spectrometer  at 25°C. The ESR spectrometer was usually set at a microwave power of 1.0 mW, amplitude of 100 kHz, and field modulation width of 0.1 mT.The 2/air at 37°C. Cells were seeded in 96-well plates with 3000 cells per well, and after overnight preincubation, the polymer conjugates P-hyd-dPyF, P-amide-PyF, or free PyF were added at different concentrations. After 24 h treatment, media were removed, and the cells were washed three times with PBS followed by replacement with fresh media. Illumination was then carried out with fluorescent blue light with peak emission at 420 nm  of 1.0 J cm-2. After further 24 h of culture, the MTT assay Mouse colon cancer C26 cells were maintained in RPMI-1640 and supplemented with 10% fetal bovine serum  under 5% CO5 cells/well and incubated overnight. Subsequently, different concentrations of P-hyd-dPyF were applied to the cells, and they were treated for 24 hours. To assess intracellular ROS, the cells were treated with the intracellular ROS probe 7-dichlorodihydrofluorescein diacetate  at a concentration of 10 μM and further cultured for 30 minutes. During this period, the esterified form of DCDFH-DA permeated the cell membranes and underwent deacetylation to form DCDHF, which could react with ROS to produce a fluorescent compound known as dichlorofluorescein. Following the incubation with the ROS probe, the cells were exposed to light irradiation as described above. Subsequently, the amount of intracellular ROS was quantitated by measuring the fluorescence intensity using flow cytometry .The C26 cancer cells were seeded in 12-well plates at a density of 2 × 10P-hyd-dPyF or free PyF were added . At the indicated time points after treatment, the media were removed, and the cells were washed three times with PBS followed by replacement with fresh media. Then, the cells were subjected to in vivo fluorescence imaging using IVIS XR  with an excitation of 675 nm and an emission of 695-770 nm. In a separate experiment, cells were seeded in 12-well pates , and after overnight preincubation, P-hyd-dPyF or P-amide-PyF were added at the concentration of 0.5 μmol . Two hours after treatment, the media were removed, and the cells were washed three times with PBS. Then, 1 mL of ethanol was added, and the cells were harvested and collected; the internalized polymer conjugates or free PyF were extracted using ethanol under sonication  on ice and quantified using fluorescence spectroscopy .The intracellular uptakes of polymer conjugates as well as free PyF were examined in the above-described C26 cells. The cells were seeded in 96-well plates . After overnight preincubation, the polymer conjugates 5) were seeded in 14 cm2 ultra-low attachment cell dishes . After six days of culture, the formed cell spheroids were transferred into 9.6 cm2 glass-bottomed culture dishes. Polymer conjugates P-hyd-dPyF, P-amide-PyF, or free PyF were then added at a final concentration of 0.5 μmol , and at the scheduled time  after treatment, the polymer conjugates or free PyF in the spheroids were visualized by confocal laser fluorescence microscopy  at the excitation wavelength of 405 nm and the emission wavelength of 570-640 nm.Further, the internalization and penetration of polymer conjugates were investigated using C26 cell spheroids. C26 cells  were purchased from SLC, Shizuoka, Japan. The animals were housed under controlled conditions of 20-22°C and 45%-50% humidity with a 12 h light/dark cycle, with free access to water and food. All of the animal experiments were approved by the Animal Ethics Committees of Sojo University  and carried out according to the Guidelines of the Laboratory Protocol of Animal Handling, Sojo University.6 cells/0.1 mL). At 10-12 days after tumor inoculation, when the tumor reached approximately 10 mm, the polymer conjugate with the concentration of 5 mg kg-1 PyF equivalent in the physiological saline solution was injected into the tail vein. The mice were sacrificed at the scheduled time, e.g., 2 h, 4 h, 8 h, 24 h, and 48 h after intravenous administration, and then, the tumor and normal tissues  were dissected and weighted. Then, DMSO was added at the concentration of 1 mL per 100 mg of tissue. The tissues were homogenized and centrifuged at 12,000 RPM at 25°C for 15 min. Moreover, an aliquot of 1 mL of blood was centrifuged at 4,000 RPM for 15 min, and then, 50 µL of the plasma was taken and diluted in 450 µL of DMSO. The quantification of the extracted PyF was performed by fluorescence spectroscopy by the dilution of supernatants in DMSO (excitation of 420 nm and emission of 650 nm). The data were then analyzed using the fluorescence intensity areas. In some experiments, the human ovarian cancer xenograft model was used, wherein human ovarian cancer A2780 cells (1 × 107) were implanted in the dorsal skin of the BALB/c nude mice . After 6-8 weeks when the tumors grew to 10-15 mm in diameter, P-hyd-dPyF was injected intravenously, and the in vivo tissue distribution assay and in vivo imaging were carried out using the same protocol as that described above. The amount of drug in each tissue was quantified using a standard curve of P-hyd-dPyF, which was added into tissue homogenates of the untreated control mice followed by the same extraction and measurement process as that described above.The mice were fed adaptively for one week before the experiments. Sarcoma S180 cells were implanted subcutaneously in the dorsal skin of ddY mice  before they were sacrificed, and the dissected tissues were subjected to fluorescence imaging by using the IVIS system and the above-described protocol (excitation of 675 nm and emission of 695-770 nm).In addition, in these experiments, the mice were subjected to P-hyd-dPyF or P-amide-PyF) was dissolved in physiological saline and administered intravenously at the indicated concentrations. The control mice were injected with physiological saline. At 24 and 48 h after the administration of the polymer conjugate, the tumor was illuminated by a xenon light  at 400-700 nm for 5 min (27 J cm-2). The tumor volume (mm3) was calculated as (W2 × L)/2 by measuring the width (W) and length (L) of the tumor, and the body weight of the mice was measured during the study period. In some experiments, with a fractionated dose of P-hyd-dPyF (2.5 mg kg-1), two cycles of PDT were performed.The S180 solid tumor model, as described above, was used in this study. At 7-10 days after tumor inoculation when the tumor diameters reached approximately 8-10 mm, each polymer conjugate (6 cells/0.1 mL) into the dorsal skin of Balb/c mice . In this study, the mice were sacrificed either 25 days after treatment or when the tumor reached a size of 2000 mm3. Subsequently, the liver and kidney were collected, and cryo-sections of 10 μm were prepared, followed by H&E staining, to evaluate the potential side effects of this treatment.The same experiment was also conducted using C26 tumor-bearing mice. The tumor model was established by injecting C26 cells , driven in the pulsed mode (~350 ns/12 kHz) by a custom-built driver, based on the iC-HG30 (iC-Haus). The NIR detection system TCMPC1270  was used with a fiber adapter and a custom-designed multi-furcated quartz fiber (Ceram Optec). The observed time window covered 80 µs, starting with excitation, at a channel width of 20 ns.To follow the tumor accumulation of the systemically injected conjugate over time using NIR detection, the animal models described in Section 2.17 were used. We investigated the t-tests were performed to assess the differences between the two groups. Analysis of variance (ANOVA) followed by the Bonferroni t-test was used for comparison between groups. The difference was considered statistically significant when P < 0.05.The results were expressed as mean ± SD. Student's P-hyd-dPyF as a tumor-targeted and pH-sensitive nanomedicine . In this study, we aimed for a specific drug release, following tumor cell uptake and reliable photosensitization in the tumor, while reducing the PS concentration in the liver and the spleen. The temporary dormant state of the PyF when bound to the polymer backbone forming self-assembled micelles is beneficial from the off-target activity point-of-view, as such a hydrophobic core-localized PyF was non-active before the disruption of the micellar systems and release from the polymer carrier .Nanomedicines exhibiting a theranostic nature are believed to be the next generation of advanced therapeutics. They can provide a real insight into the treatment's procedure and thus increase the therapy efficacy in real time by the modification of the dosing. Recently, we reported HPMA-based theranostics-bearing PyF bound by stable amide bonds and proved both the imaging potency and the PDT effect of these systems, which also showed prolonged circulation times and tumor-targeted accumulation based on the EPR effect P1, poly(HPMA-co-MA-AH-NHNH2), with hydrazide groups statistically distributed along the polymer chain showed molecular weights around 26⋅103 g mol-1 and a dispersity below 1.05. Similarly, the linear polymer precursor P2, poly(HPMA-co-MA-APMA), with amine groups showed molecular weights around 30⋅103 g mol-1 and a narrow dispersity (Table 1). In both of the cases, the number of functional groups was adjusted to be sufficient for the further attachment of the active PS moiety. P1 hydrazide groups allowed the attachment of the dPyF up to 7 wt.%, forming the polymer conjugate P-hyd-dPyF with dPyF bound via the hydrolytically cleavable and pH-sensitive hydrazone bonds , and the control polymer conjugate P-amide-PyF was prepared from the precursor P2 by the attachment of the PyF via an amide bond as described recently The controlled RAFT polymerization technique was used to synthesize almost monodisperse polymer precursors with molecular weights below the limit of glomerular filtration P1 and P2 showed the typical DH around 9-10 nm for the linear HPMA-based polymer random coils in aqueous solutions, ensuring their safe elimination from the organism via renal filtration after the drug release ). Indeed, the attachment of PyF or its derivative dPyf to the polymer carrier significantly increased the hydrodynamic sizes for the polymer-PyF/dPyF conjugates in an aqueous solution, DH ≈ 15 nm, indicating the macromolecules' self-assembly into micelles due to the amphiphilic character caused by the presence of the hydrophobic PS in their structures (Table 2). The TEM image also proved the micelle formation of P-hyd-dPyF showing a diameter of about 15 nm , which agreed with the SEC results. Indeed, the conjugate P-hyd-dPyF showed a very small fraction of unimers, less than 5%, indicating a predominant formation of stable micelles. The small amount of unimers can be ascribed to the presence of polymer chains without a dPyF molecule . For the conjugates P-hyd-dPyF and P-amide-PyF no significant heat signature, which can be attributed to the dissociation of porphyrin stacks upon dilution was found until the concentration of 0.011 mg.mL-1 (-1 (detection limit of the method). We can conclude that both the polymer conjugates form a stable micelle, which CMC is below 0.011 mg. mL-1.As the hydrodynamic sizes of both polymer micelles were above the renal filtration threshold, their reduced urinary excretion and prolonged circulation in the organism could be expected, which is an advantage from the pharmacokinetics point of view. Moreover, the characteristics of these amphiphilic systems were additionally evaluated using FFF. In the case of precursor  mg.mL-1 .5. AlthoP-hyd-dPyF in the absence/presence of different compounds, such as Tween-20, SDS, lecithin, and urea . In aqueous solution , we observed almost no fluorescence, indicating fluorescence quenching and suggesting the micelle formation of P-hyd-dPyF. However, in the presence of surfactants such as Tween-20 and SDS, which have the capability to disrupt micelle self-assembly, we observed strong fluorescence. Additionally, incubating the samples with urea resulted in no fluorescence. These findings indicate that hydrophobic interactions, rather than hydrogen bonds, play a crucial role in the micelle formation. Importantly, when lecithin, a lipid component found on cell membranes, was added, a significant increase in fluorescence was observed. This suggests that the micelle formation of P-hyd-dPyF is disrupted during intracellular uptake. These results provide valuable insights into the behavior of P-hyd-dPyF in different environments and in vivo scenarios. The disruption of micelle formation in the presence of specific surfactants and lecithin highlights the importance of understanding the interactions and behavior of P-hyd-dPyF in various biological settings.We also investigated the fluorescence from the conjugate P‑hyd-dPyF increased by approximately eight times when Tween-20 was used at 0.5% (v/v), which was similar to the results published previously P-amide-PyF and increased by fourteen times when SDS was used at 0.3% (w/v). Taken together, these findings can be correlated with the lower generation of 1O2 in PBS even after illumination and the higher generation of 1O2 after the micelle disruption by Tween-20 , ensuring the safety of the micellar systems in circulation. Moreover, the attachment of PyF or dPyF to the polymer carriers decreased the fluorescence intensity in EtOH and almost depleted the intensity in PBS due to the excitonic interactions between PyF molecules distributed along the polymer backbone, confirming the tendency of PyF or dPyF to stack together and to form the hydrophobic core of micelles or aggregates in aqueous solutions . We observed a similar behavior for the UV-Vis absorption spectrum, which indicates the depletion of absorption after the attachment of PyF to the polymer carrier ). More importantly, when P-hyd-dPyF was dissolved in PBS at pH 5.0 with SDS as the surfactant, the fluorescence increased to almost the same as that found for the PyF compound, which can be ascribed to the PyF release from the conjugate , whereas P-amide-PyF showed much lower fluorescence at the same condition . Such behavior may be highly important in the tumor microenvironment conditions in vivo. We hypothesized that, particularly for the P-hyd-dPyF conjugate investigated here where the polymer backbone carries several dPyF moieties, the release of dPyF in the tumor area could increase the fluorescence and possibly the singlet oxygen production.The fluorescence of 1O2 generation from P-hyd-dPyF was suppressed in PBS at pH values of 6.5 and 7.4 even after 450 s of illumination, confirming the conjugate self-assembly and micelle formation in aqueous solutions. The 1O2 generation was depleted in the same manner as the fluorescence. We considered this an advantage, as the aggregation and thus the low activity of dPyF is highly welcome during circulation and transport to the tumor tissue. However, once in the tumor, the generation of 1O2 is necessary for effective PDT therapy; therefore, we diluted P-hyd-dPyF in a solution of Tween-20 in PBS 7.4 for comparison . Clearly, the addition of Tween-20, amphiphilic surfactant, increased the 1O2 generation dose-dependently, which was consistent with the fluorescence increase observed before. Here, the Tween-20 could separate the aggregated dPyF molecules into Tween-20-dPyF nanovesicles, leading to the disintegration of the polymer-based micelles, thus stopping the excitonic interaction and increasing the 1O2 generation. More importantly, we found a further increase of 1O2 generation at pH 5.0 in the presence of Tween-20 , which supported our hypothesis that the restoration of the 1O2 generation in the tumor tissue would be based on the disassembly of the micellar structures below the critical micellar concentration and the subsequent release of dPyF from the polymer conjugate and/or embedding into membranes in the tumorous tissue. In summary, the present P‑hyd-dPyF conjugate had to be safe and non-effective during the delivery, showing no or negligible activity, and the final therapeutic activity could be restored because of the micellar disruption and/or the release of freely active dPyF. Just after dissolution of the P‑hyd-dPyF, the quantum yield of singlet oxygen (∆φ) in the presence of surfactant (0.1% SDS) reached for all studied pH  value 0.5. This is highly comparable with free PyF, which shoved value of 0.52. Importantly, the P-amide-PyF had the value much lower (0.31), thus showing the benefit of the structure of novel P‑hyd-dPyF conjugate.We found that the P-hyd-dPyF was evaluated at 37°C in pH 5.0 and 7.4, modeling the acidic lysosome environment of the tumor cells, where the conjugates should be located after the cellular uptake, and the typical neutral blood conditions, respectively. The pH-dependent drug release was enabled by attaching dPyF to the polymer carrier via hydrazone bonds, which were quickly hydrolyzed at pH = 5.0, releasing up to 90% of dPyF after 24 h, in comparison with 30% of dPyF released at pH = 7.4 . The conjugate fulfills the basic criteria for a pH-sensitive pro-drug, relatively stable in the blood circulation and the fast dPyF release in the tumor tissue. The P-amide-Pyf conjugate was also incubated in both the buffers and no release of Pyf within 5 days was observed.The dPyF release from the conjugate P-hyd-dPyF, and suggest its superior physiochemical characteristics, particularly the pH-responsive release profile compared with P-amide-PyF. We thus anticipated the superior PDT effect of P-hyd-dPyF and first examined its intracellular uptake dynamics by using cultured colon cancer C26 cells as well as the three-dimensional cell culture models, i.e., spheroids.The above-described results showed the nano-micellar formulation of P-hyd-dPyF or free PyF, and the internalized drugs were visualized by detecting the fluorescence originated from PyF and semi-quantified by using the fluorescence intensity. P-hyd-dPyF showed a time-dependent intracellular uptake as the increase in the internalized drug continued up to at least 24 h . In comparison, free PyF showed a similar but considerably faster intracellular uptake as the fluorescence originated from PyF reached a peak at 4-8 h after addition to the cells followed by the decrease in the fluorescence, which may suggest the degradation or beginning of the excitonic interaction due to the high local concentration Figure ). More importantly, compared with P-amide-PyF, P-hyd-dPyF exhibited a significantly higher intracellular uptake, which was evaluated after 2 h of treatment and quantified by fluorescence . Here, we assumed that there was a concurrence of the internalization of dPyF released outside the tumor cells and P-hyd-dPyF itself. These findings suggest that the release of free dPyF is an important step for its efficient cell internalization, thus fulfilling the therapeutic effect. Moreover, the internalization of dPyF using P-hyd-dPyF was comparable to free PyF, showing the huge advantage of such stimuli-responsive nanomedicine already in vitro. We also expected the slower intracellular uptake profile of P-hyd-dPyF than that of free PyF as the dPyF release from the polymer conjugate is a time-dependent event. We further examined the behavior of polymer conjugates versus that of free PyF by using an in vitro 3D C26 spheroid model and confocal microscopy . The uptake of free PyF by spheroids was clearly observed 4 h after the treatment and then gradually decreased similarly, as shown in the cultured C26 cells . In parallel with the results shown in Figure P-hyd-dPyF exhibited a comparable but slower uptake than free PyF, whereas no visible fluorescence appeared up to 8 h after the P-amide-PyF treatment . These findings reveal the superior uptake property of P-hyd-dPyF to that of P-amide-PyF. We hypothesized that the beneficial uptake of P-hyd-dPyF could be linked to the enhancement of the therapeutic effect. Importantly, Figure Figure ).Cultured C26 cells were treated with P-hyd-dPyF, we first evaluated its cytotoxicity in the cultured C26 colon cells in the dark or under illumination. For the assay of dark cytotoxicity, the cells were treated for 48 h in the dark, and for PDT, after 24 h of treatment in the dark, illumination using a blue light source with the maximum absorption of 420 nm was carried out (1 J cm-2), followed by further 24 h incubation in the dark. In our previous study using P-amide-PyF, we found red light irradiation (680 nm) showed neglectable PDT effect compared to blue light exposure, suggesting the light corresponding to the maximum absorbance of PyF is most suitable for PDT using PyF To investigate the PDT effect of P-hyd-dPyF showed no apparent cytotoxicity up to 0.5 μmol in the medium. Instead, after illumination using the blue light source, their cytotoxicity remarkably increased in a dose-dependent manner: all of the cells died at 0.1 μmol. More interestingly, the PDT effect of P-hyd-dPyF was comparable and even stronger than that of the free PyF , probably because of the accumulative internalization profile of P-hyd-dPyF: the intracellular uptake of P-hyd-dPyF increased time-dependently to at least 24 h, whereas the internalization of free PyF reached the peak at 4-8 h after which it decreased . Moreover, we evaluated the conjugates' cytotoxicity and observed a considerably stronger PDT effect of P-hyd-dPyF (IC50 of approximately 0.03 μmol) than that of P-amide-PyF (IC50 of approximately 0.6 μmol) . These findings again support that the internalization and rapid release of free dPyF plays a critical role in the PDT effect of PyF conjugates: P-hyd-dPyF thus exhibited a superior PDT effect to that of P-amide-PyF because of the cleavable hydrazone bond and the preferable release profile .The cell viability was examined using MTT assay. In the dark, free PyF and P-hyd-PyF, we conducted measurements of intracellular ROS generation during PDT using P-hyd-PyF. The results revealed a dose-dependent increase in intracellular ROS levels . These findings provide additional support for the notion that the cytotoxicity of P-hyd-PyF is attributed to the PDT effect, specifically the generation of ROS.To further confirm the PDT effect of P-hyd-dPyF is how the pH-stimuli sensitiveness would affect the in vivo behavior. We thus investigated the in vivo tissue distribution dynamics of P-hyd-dPyF by injecting the conjugate at 5 mg kg-1  in S180 tumor-bearing mice when the tumor grew to approximately 10 mm in diameter. Figures in vivo imaging exhibited an extensive fluorescence in the tumor with no or very low background fluorescence, which correlated with the semi-quantification of the drug accumulated in the tumor. Moreover, we observed a strong fluorescence at 2 h after the intravenous injection, and these high levels were maintained in the tumor to at least 12 h, and then gradually decreased; however, even at 48 h after the intravenous injection, a comparable strong fluorescence signal could still be detected in the tumor . In parallel, P-hyd-dPyF maintained a relatively high concentration in the plasma, with the plasma half-life > 12 h . These results clearly reveal that P-hyd-dPyF behaves as a nanomedicine in vivo showing a prolonged circulation time and tumor selective accumulation by taking advantage of the EPR effect.One concern regarding P-hyd-dPyF in different tissues, we found a high plasma concentration along with a high drug accumulation in the tumor up to 8 h after the intravenous injection. Moreover, comparable levels of the drug were found in many major organs  . However, more importantly, at 24 h after the intravenous injection, the amount of drug in the normal tissues considerably decreased, whereas a high level in the tumor was maintained, resulting in a tumor/liver drug ratio of 20; i.e., the released dPyF in the tumor was 20 times higher than that in the liver . Notably, almost no drug was found in the normal tissues after 48 h, whereas a high level in the tumor still remained . We also observed similar results for the fluorescence imaging of dissected tissues . Importantly, the tissue distribution for P-hyd-dPyF was completely different in comparison to P-amide-PyFP-amide-PyF was after 24 h predominantly localized in the liver and to lesser extent in spleen, plasma and tumor. We summarize that the stimuli-sensitive P-hyd-dPyF enable to enhanced the PyF biodistribution with predominant tumor accumulation and low off-target localization.After the mice were sacrificed, tumors and normal tissues were collected, and dPyF was extracted to measure fluorescence spectroscopy. When we examined the distribution of P-hyd-dPyF was confirmed by using a human ovarian cancer xenograft model , and the quantification analysis showed that the tumor concentration of P-hyd-dPyF was approximately 14% of the injected drug/g tissue . These findings indicated the beneficial tissue distribution dynamics of P-hyd-dPyF, which not only exhibited a sustained and targeted accumulation in the liver but also could achieve an extremely high tumor/liver ratio that is hardly seen in many other nanomedicines, particularly nano photosensitizers. In fact, these nanomedicines could be taken up by the reticuloendothelial system in the liver and spleen, P-hyd-dPyF as the released dPyF was excreted from urine or from bile after metabolization in the liver. This notion was supported by the in vivo distribution profile of P-hyd-dPyF, which showed relative high levels of the drug in the kidney even after 24 h , and the fluorescence in the liver at 4 h after the intravenous injection was found mostly in the gall bladder . However, polymer conjugates and the released highly hydrophobic dPyF could not be cleared from the tumor efficiently because of the poor lymphatic function; P-hyd-dPyF, a rapid release of dPyF could be attained in the weak acidic condition of solid tumors, This body distribution profile of in vivo imaging of P-hyd-dPyF showed a clear fluorescence signal in the tumor with very low contrast background signal . One important advantage of PyF is that it can be excited using the light at 670 nm in which the long wavelength ensures the deep penetration into the tissues and that it can emit fluorescence with a wavelength longer than 700 nm that facilitates the detection with less interference of autofluorescence. Using this Ex/Em combination, we detected the tumor with high selectivity and specificity as supported by the results shown in Figures exc = 670 nm that served only for diagnosis, because no 1O2 was produced together with a high contrast of the tumor tissue (lower fluorescence of the polymer conjugate and higher fluorescence of released dPyF). Instead, in our experiments, illumination at λexc = 420 nm reached a considerably better therapeutic effect as a result of the 1O2 generation, even though the penetration depth of blue light is very limited. Based on previous results, The P-hyd-dPyF may become a potential candidate drug for this therapeutic approach because of its high tumor selectivity and superior in vivo imaging property, which warrants further investigation.Moreover, we are aware that the tumor location is important as PDT can only treat tumors where light can reach. Besides superficial tumors with easy access to light, it is possible to apply interstitial, endoscopic, or laparoscopic devices for the delivery of light into more difficult access treatment sites. Unfortunately, solo therapy using PDT cannot be applied to treat metastatic tumors or patients with allergies to these compounds in vivo PDT effect of P-hyd-dPyF by using the mouse sarcoma S180 solid tumor model. Because of the highly tumor selective accumulation demonstrated above, we expected the superior therapeutic effect of P-hyd-dPyF. According to the tissue dynamics of P-hyd-dPyF , we carried out the illumination by using a xenon light source for 5 min at 24 h and 48 h after the conjugates' administration; when P-hyd-dPyF was delivered and retained in the tumor, whereas no or very few remained in normal tissues. Moreover, this time setting ensured the dPyF release that further increased the PDT efficacy. The illumination was carried out with a broad wavelength light of 400-700 nm, which could ensure sufficient illumination intensity while simultaneously covering the appropriate light range  to efficiently excite PyF. Consequently, this strategy enhanced the therapeutic outcome of cancer treatment when compared with the conventional chemotherapy.Finally, we investigated the P-hyd-dPyF at 5 mg kg-1 , and the effect was considerably superior to that of the PDT using P-amide-PyF, the tumor inhibitory rate of P-hyd-dPyF and P-amide-PyF at day 6 after treatment was 64.2% and 39.1%, respectively . However, when we further investigated the optimal dosing and therapeutic regimen, unexpectedly and interestingly, we found that the lower dose of P-hyd-dPyF  showed a considerably better therapeutic effect than the higher doses , and almost a complete inhibition of tumor growth was observed up to 18 days after the treatment . Similarly, the two-round PDT using 5 mg kg-1 of P-hyd-dPyF exhibited similar results to those of the one-round PDT of 10 mg kg-1P-hyd-dPyF, both of which showed a decreased therapeutic effect than that of the one-round of 5 mg kg-1P-hyd-dPyF . Although it is not completely clear why P-hyd-dPyF exhibited the reversed dose-dependency, potential explanations include the following: i) the relatively low dose of 2.5 mg kg-1 was sufficient to exert a sufficient PDT effect, mostly because of the tumor selective accumulation and retention of P-hyd-dPyF, and ii) higher doses of P-hyd-dPyF were not preferable for PDT. Regarding the later issue, a recent study from our group indicated that very intense illumination during PDT negatively influenced the 1O2 generation and the PDT effect, in which photosensitization and oxygen consumption exceeded the oxygen supply, limiting the generation of 1O2 to blood vessels and their direct vicinity, whereas less 1O2 was generated in the surrounding tumor tissues P-hyd-dPyF, which triggered a rapid and vast generation of 1O2 mostly in blood vessels but without the sufficient PDT effect in the tumor tissues because of the lack of oxygen. Moreover, 1O2 and ROS generated in the blood vessels would destroy them, further exacerbating the hypoxic or anoxic condition, consequently influencing the PDT effect. It is thus reasonable to use a relative low dose of P-hyd-dPyF and moderate intensity of light as the optimal PDT regimen. A future follow-up study will focus on this issue to clarify the influence of dosing/illumination balance on the PDT effect as well as the fluorescence imaging efficacy, towards the clinical development of P-hyd-dPyF as a theranostic agent.As expected, we achieved a significant tumor growth suppression by one round of PDT using P-hyd-dPyF the in vivo PDT effect of both polymer conjugates was evaluated by using the mouse colon cancer C26 solid tumor model, see . Importantly, we have found similar superior treatment efficacy of P-hyd-dPyF as in the case of mouse sarcoma model. Also, the reverse dose dependency was observed showing excellent treatment efficacy of the stimuli sensitive theranostics P-hyd-dPyF. In addition, during the PDT treatment using P-hyd-dPyF as described above, no apparent body weight loss  and no apparent inflammatory changes (reddish and blackish coloration) in the skin were observed, not only in the skin away from the tumor but also in the skin surrounding the tumor, which was illuminated during the treatment. Histological examination (H&E) staining also showed no apparent histological changes in the major organs, i.e., the liver and kidney, induced by the treatment . These findings suggest the safety of the P-hyd-dPyF-based PDT.To verify the beneficial therapeutic effect of in vivo without harming the animal. Such a method would allow considerably better supervision while reducing the number of required animals. The effect that had to be exploited for this quantification in the tumor tissue, was the PDT-induced anoxia, which was described previously -2, which was clearly above the estimated threshold for this effect to occur. Figure P-hyd-dPyF. The optical fiber was positioned centrally on the tumor with direct skin contact. It was quite obvious that there were huge differences for the investigated tumors in the time region of 0 to 30 µs after excitation. Following our previous explanation, we assigned these signal components to the singlet oxygen that was generated by such a drug that was still circulating in the blood vessels. In the blood flow, there was no lack of oxygen and the signal components showed the typical double exponential behavior, masked by the strong short-term artifact of such measurements. Such signals could be detected for days all over the body of the mouse after the drug injection. However, the intensity varied considerably with the location, which was no surprise as the number of blood vessels and their size varied as well. This also applied, of course, to blood vessels that were near or in the tumor, which contributed to the detected signal. In principle, such direct observation of the generated singlet oxygen would be an option to quantify the drug concentration in blood, if we managed to assure the better reproducibility of the positioning. The point here, however, was that the signals from the dPyF in the blood decayed a few tens of microseconds after excitation, while in the time window of 40-80 µs, we mainly detected the phosphorescence of the extravasated drug. For tumors of similar size (5-8 mm) and identical applied drug amounts, this signal component showed little variation. As we expected immediate anoxia in the tumor after illumination, the PyF phosphorescence intensity had to be, in the first approximation, proportional to the local PyF concentration in the tissue. This measurement was considerably robust against the misplacement of the fiber tip, as the tumors were bigger than the optical fiber and because the signal scattering was average over several cubic millimeters of the tumor.We tested whether the accumulation of the polymer conjugate or better, the accumulation of active dPyF, could be optically quantified P-hyd-dPyF was the highest just 6 h after injection and decayed slowly. This corresponds to our other results and demonstrates that time-resolved phosphorescence detection is an appropriate method to determine the amount of active dPyF in the tumor.The measurements were repeated at several time points after injection. Figure exc = 670 nm. The beneficial tissue distribution dynamics of the tumor microenvironment-responsive system also resulted in a superior PDT effect because of the specific PyF release and activation of its theranostic nature in the acidic tumor environment. Once in the tumor, illumination at λexc = 420 nm allowed a remarkable antitumor PDT effect in the mouse sarcoma S180 solid tumor model. Indeed, low doses of PyF proved to be optimal for superior therapeutic effects that resulted from the 1O2 generation. We are convinced that such theranostic that is functional at low doses is an excellent candidate for further preclinical and clinical development.Herein, we successfully developed and deeply evaluated polymer-based nanomedicines serving as stimuli-responsive theranostics in the treatment and visualization of solid tumors. PyF bound by the pH-sensitive hydrolyzable hydrazone bond to the polymer carrier enabled the tumor site-associated simultaneous PDT and tumor imaging. The safety of such theranostics in circulation was ensured by their self-assembly into micellar structures in aqueous solutions. Moreover, the stimulus-sensitivity behavior showed beneficial properties for the tumor-targeted delivery, reducing their accumulation in the liver and spleen. The nanomedicine was stable during the transport to the tumor tissue, and after the micelles' selective accumulation in solid tumors via the EPR effect, their disruption was confirmed by the appearance of a strong fluorescence signal with almost no background at λSupplementary figures.Click here for additional data file.


As authors of the text, we can only thank Mattei et al. . for theThey expressed concern regarding the definition of JD that we reported. In particular, the concepts of pseudo-joint and the involvement of a morphological change in the coronoid process were identified, which seems to be truly a big jumble deepening this topic. Their reply tries to better explain that the original definition of Jacob’s disease does not report any reference to an abnormal coronoid process in the mandible, stating as Octave Jacob described “a coronoid process of normal shape and size associated with a hyperostosis of the posterior part of the zygomatic bone . The pos


In an LPS-stimulated ALI animal model, pharmacologicalsEH inhibition by WED or sEH knockout (KO) alleviated pulmonary damage,such as the increase in the alveolar wall thickness and collapse.Additionally, WED or sEH genetic KO both suppressed macrophage activationand attenuated inflammation and oxidative stress in vivo. These findings provided the broader prospects for ALI treatmentby targeting sEH to alleviate inflammation and oxidative stress andsuggested WED as a natural lead candidate for the development of novelsynthetic sEH inhibitors.Soluble epoxide hydrolase (sEH) plays a critical rolein inflammationby modulating levels of epoxyeicosatrienoic acids (EETs) and otherepoxy fatty acids (EpFAs). Here, we investigate the possible roleof sEH in lipopolysaccharide (LPS)-mediated macrophage activationand acute lung injury (ALI). In this study, we found that a smallmolecule, wedelolactone (WED), targeted sEH and led to macrophageinactivation. Through the molecular interaction with amino acids Phe362and Gln384, WED suppressed sEH activity to enhance levels of EETs,thus attenuating inflammation and oxidative stress by regulating glycogensynthase kinase 3beta (GSK3β)-mediated nuclear factor-kappaB (NF-κB) and nuclear factor E2-related factor 2 (Nrf2) pathways  Targeting sEHwith WED to enhance levels of EETs suppressedthe macrophage activation via regulating GSK3β-mediated NF-κBand Nrf2 pathways, which resulted in the attenuation of LPS-inducedALI. Recently, sEH inhibition to enhancelevels of EETs has become an attractive research strategy to treatdiseases related to inflammation, such as lung injury and diabetes.26Polyunsaturated fatty acids (PUFAs)belong to a number of ω-3and ω-6 families and play an important role for maintainingthe physiological health through their bioactive metabolites. Thebest studied of the polyunsaturated fatty acids is arachidonic acid(AA) and other PUFAs.28 Increasing evidence supports the therapeutic effects of naturalproducts and traditional Chinese medicines, such as kuraninone, alisolB, -britanicafanin A, 3β-hydroxy-25-anhydro-alisolF, and Inula japonica.31 Wedelolactone (WED), first isolatedfrom Wedelia calendulacea by Govindachari and co-workersin 1956, is a polyphenol sharing a coumarin skeleton with a benzofuranmoiety at C-3 and C-4.32 Emerging evidencedemonstrates multiple pharmacological responses of WED, includinganti-inflammatory, anticancer, and hepatoprotective effects, as wellas the remission of Parkinson’s disease (PD) and kidney injury.37 Recent research focused on WED has indicated that it attenuateslung collagen deposition and fibrotic pathology in bleomycin-mediatedpulmonary fibrosis.38 Similarly, WED protectsbronchial epithelial cells from cigarette smoke extract-induced damage,as well.39 However, the protective mechanismand molecular target of WED in macrophage-mediated respiratory diseases,especially acute lung injury (ALI), still remains to be elucidated.An effective approach to discover innovative drugs involvesprobingnatural products for possible drugs or leads because of their complexand changeable structures and multiple biological activities.Herein, we investigated the ability of WED to reduce inflammationand oxidative stress in LPS-activated macrophages. To understand howit exerted the effect, we performed the target fishing experimentson the basis of the affinity chromatography to identify tentativelythe cellular direct target of WED. We found that targeting sEH withWED enhanced levels of EETs to trigger the glycogen synthase kinase3beta (GSK3β) inhibition, thereby leading to modulation of thenuclear factor-kappa B (NF-κB) and nuclear factor E2-relatedfactor 2 (Nrf2) pathways. Meanwhile, pharmacological sEH inhibitionby WED or sEH knockout (KO) exerted a significant therapeutic effectin the ALI animal model treated with LPS. Collectively, this studyrevealed that sEH served as a valuable target for the treatment ofthe inflammatory response and oxidative stress of ALI.Figure S1). As described in Figure S1). Furthermore,WED reversed the upregulation of LPS-induced inflammatory genes , and proteins  through the NF-κBpathway in a dose-dependent manner  in LPS-induced RAW264.7 macrophages asthe cytotoxicity of 40 μM of WED , thereby requiringthe protective effect of WED toward LPS-mediated mitochondrial damage.Nrf2 is the key transcript factor involved in the mitochondrial redoxsystem; thus, we also assayed for the regulatory effect of WED towardthis pathway. Pretreatment of WED  upregulatedexpressions of heme oxygenase-1 (HO-1), glutamate–cysteineligase catalytic subunit (GCLC), glutamate–cysteine ligasemodifier subunit (GCLM), NAD(P)H/quinone oxidoreductase 1 (NQO-1),and Nrf2; downregulated the kelch-like ECH-associated protein l (Keap1)expression; and regulated mRNA levels of their corresponding genes,such as HO-1, Keap1, NQO-1, and Nrf2, in LPS-induced macrophages (S3). Additionally, WED treatment promoted thetraffic of Nrf2 to the nucleus in LPS-induced macrophages .The TNF-α staining results suggestthat WED pretreatment  suppressed the increaseof TNF-α positive cells in lungs of LPS-induced ALI mice 4A, as weNA level 4B. MoreoS4B revealed that the LPS challengeled to a striking increase in the malondialdehyde (MDA) level andthe mtDNA content and a remarkable decrease in the levels of GSH andSOD, which were significantly suppressed after WED treatment . The mitochondrial architecture in the control group(S5). Additionally, WED treatment reversed LPS-mediatedoxidative stress in vivo by activating the Nrf2 pathway,which was supported by the experiments on the basis of the Nrf2 staining,Western blot, and PCR (S6). These aforementionedresults revealed anti-inflammatory and antioxidant effects of WED in vivo.The ROS imbalance leads to oxidative stress, whichcontributesto the mtDNA damage in the ALI and leads to the abnormal mitochondrialultrastructure. ol group4D was chol group4D. The aol group4G and S5Kd values of 87.2 and 68.7 nM (S9). Inaddition, WED enhanced the level of the sEH substrate 14,15-EET andreduced its diol level in LPS-induced macrophages . In addition, sEH rescue aggravated LPS-inducedinflammation and oxidative stress. This was illustrated by expressionsof phosphorylated p65, Nrf2, and their downstream genes (S10), while the protective effect of WED in vitro after sEH rescue was significantly weakened (S10). These results demonstrate the effects ofWED depend on its interaction with the sEH target.Next, we performedsEH knockdown and rescue experiments to explore the role of sEH inanti-inflammatory and antioxidant effects of WED in LPS-stimulatedmacrophages. As shown in and Nrf2 6A,C. Knoand Nrf2 6B, whereS11). The root-mean-square deviation(RMSD) of the WED–sEH complex remained about 1.5 Å  during the 40 ns of the molecular dynamic stimulation, revealingthe stability of the WED–sEH complex. The protein trajectoriesof apo-sEH and the WED–sEH complex revealed that the bindingof WED with sEH reduced the volume of the catalytic cavity of sEH. There were still about 1–4 hydrogenbonds between WED and Phe362, Ile363, Ser374, Asn378, Tyr383, Gln384,Gln502, and Met503 in the molecular dynamic stimulation , thereby allowingthe inhibition of the GSK3β activity (S12A). Similarly,inhibition of sEH by WED could decrease the GSK3β activity inmacrophages after LPS exposure (S12C) whileremarkably increasing the GSK3β activity after sEH rescue (S12B). Therefore, we further investigated whetherthe downstream key pathway involving the sEH was involved in the protectiveeffect of WED using the GSK3β inhibitor LiCl. Inhibition ofGSK3β by LiCl (5 mM) attenuated macrophage-activation-mediatedinflammation and oxidative stress, such as expressions of phosphorylatedp65, Nrf2, and their downstream genes . These results suggest that the anti-inflammatoryand antioxidant effects of WED depend on sEH to regulate the GSK3β-mediatedNF-κB and Nrf2 pathways through EETs in macrophages.Accumulating evidence hasdemonstrated that the inhibition of sEH suppresses the GSK3βregulation of the NF-κB and Nrf2 pathways in the nervous system.am genes 8D,E. It am genes 8D,E. NexFigure S14).Next, we found that WED treatment significantly inhibited the decreaseof levels of sEH substrates, such as 11,12-EET and 14,15-EET, exceptfor in the case of low dose WED treatment . Conversely, the effect of WED toward 8,9-EET was notsignificant in LPS-exposed ALI mice except for the high dose of WED. As expected, administrationof WED remarkably decreased levels of the corresponding diols, including8,9-DHET, 11,12-DHET, and 14,15-DHET. Notably, WED treatment suppressedthe sEH catalytic activity on the basis of the ratio of EETs and theircorresponding DHETs  mice through the deletion of four exonsof sEH from exon 2 to exon 5, which were supported by the genotypingand Western blot analysis . In addition, Ephx2 geneticKO resulted in an increase in HO-1 and Nrf2 positive cells (S16B). Further effects were not observed in LPS-inducedALI Ephx2–/– mice treatedwith WED. These results are consistent with anti-inflammatory andantioxidant effects of WED being dependent on sEH inhibition.Lastly, the effects of WED on LPS-induced inflammationand oxidative stress were investigated in ve cells11A and ive cells 11D and ive cells 11E. The ve cells 11E depen24 In this study, sEH was identifiedas a key target for ALI by enhancing levels of EETs to suppress themacrophage activation. Moreover, we described the mechanistic insightsinto the sEH inhibition by the small natural molecule WED by targetingamino acid residues Phe362 and Gln384. Additionally, we demonstratedsEH as an important modulator of the GSK3β-mediated NF-κBand Nrf2 pathways in macrophages for inflammation and oxidative stress.These findings provided broader prospects for the treatment of ALIby targeting sEH to inhibit the macrophage activation and suggestedWED as a potential agent for the development of sEH inhibitors.The C-terminal hydrolase activity of sEHis in a family of α/βhydrolase proteins. The inhibition of this C-terminal domain stabilizeslevels of EETs to regulate various biological processes; therefore,sEH functions as a promising therapeutic target for diseases relatedto inflammation.3 Depending on the pathogen recognition receptors, macrophages canphagocytize bacteria and other pathogens and inhaled particulates,especially in the respiratory system, meanwhile producing abundantinflammatory mediators,42 such as TNF-α and IL-6. This brings about mitochondria dysfunctionvia effects on the expression of proteins involved in the fusion andfission,8 such as Mfn1,Opa1, Drp1, and Fis1. Furthermore, the mitochondria release an amountof ROS, which contributes to oxidative stress through the inactivationof the Nrf2 pathway. Previous studies have demonstrated the effectof WED toward inflammation and oxidative stress in Parkinson’sdisease and kidney injury.37 Furthermore, WED has attenuated bleomycin-mediated pulmonary fibrosisand protected bronchial epithelial cells from cigarette-smoke-extract-induceddamage,39 which suggests the potential of WED againstinflammation and oxidative stress in ALI. As our expected, these phenomenawere all observed by regulating the NF-κB and Nrf2 pathwaysin LPS-induced macrophages and ALI animals after WED treatment.Macrophages are cellular components of the innate immune systemthat reside in virtually all tissues and possess the capacity forcleaning apoptotic cells and releasing growth factors, thereby contributingto inflammation, innate immune response, and homeostasis.43 Recent researcheshave reported the application of the affinity chromatography, biotin–avidin,and ligand-induced stability shift-dependent proteomics technologiesfor discovering the direct target of natural products, such as kurarinone,alisol B, eupalinolide B, and handelin.45 In this study, we uncovered that sEH was the directcellular target of WED on the basis of technologies employing affinitychromatography. The sEH is a bifunctional enzyme with the C-terminalhydrolase and N-terminal phosphatase activities. The sEH is responsiblefor the hydrolysis of the bioactive metabolites EETs transformed byCYP2J, CYP2C, and other CYP enzymes from AA to their correspondingepoxides.23 Increasing evidence has revealed the roleof sEH in inflammation and oxidative stress-mediated lung diseases.48 For example, chemical inhibition of sEH by 1-trifluoromethoxyphenyl-3-(1-propionylpiperidin-4-yl)urea (TPPU) suppressed LPS-mediated macrophage activation in vitro to regulate the proinflammatory cytokine level,51 increased the survival rate of LPS-induced ALI, and attenuated theneutrophil infiltration and alveolar capillary leakage in the ALIanimal model.50 Furthermore, Ephx2 genetic KO attenuated the pulmonary inflammation and edema in hyperoxia-or cigarette-smoke-mediated lung injury mice,48 thereby suggestingthe potential of sEH in macrophage-activation-induced ALI. As expected,sEH knockdown alleviated the inflammation and oxidative stress, andthe sEH rescue magnified the abovementioned changes in vitro. Similarly, sEH KO remarkably attenuated the course of ALI in anLPS-induced mouse model. Furthermore, the protective effect of WEDwas abolished by sEH knockdown, KO, and rescue.Although the ability to define drug target protein recognitiontechnology is rapidly developing, the discovery of drug targets stillfaces many challenges.52 They are all slow, tightly binding competitiveinhibitors based on their urea or amide moieties because of theirhydrogen bond interaction with amino acid residues Asp333, Tyr381,and Tyr465, in charge of opening and fixating on the epoxide ring,respectively.17 Recently, we have reportedan uncompetitive sEH inhibitor kurarinone with an anti-PD effect andrevealed that it interacted with Tyr343, Ile363, Gln384, and Asn472in the surrounding of the catalytic cavity according to its cocrystalwith sEH.24 Similarly, WED suppressed thesEH activity through hydrogen bonds with Phe362 and Gln384, whichwas supported by the results of the pull-down, CETSA, and DARTS experimentsthrough Phe362Ala and Gln384Ala mutations.Several sEHinhibitors, such as the ureas AR9281 (UC1153), EC5026, and GSK2188931B (an amide), have entered human safetytrials.in vitro and in vivo. Furthermore, we identified small moleculeWED as an inhibitor for targeting sEH through the interaction withamino acid residues Phe362 and Gln384. These findings provid broaderprospects for the ALI treatment by targeting sEH to alleviate theinflammation and oxidative stress and suggest WED as a natural productdrug and a leading candidate for the development of new sEH inhibitors.Collectively, wefirst reported that sEH inhibition served as atarget for the treatment of ALI through enhancing the EETs level toregulate GSK3β-mediated NF-κB and Nrf2 pathways, whichresulted in the inactivation of macrophages Inula britannica on the basis of the 1H and 13C NMR spectra  with support of a check of purity by thin-layer chromatographyand purity and structure by LC-MS. The primary antibodies for sEH(10833-1-AP), NQO-1 (A1518), GSK3β (A2081), p-GSK3β (5558),TNF-α (17590-1-AP), IL-6 (21865-1-AP), iNOS (13120), MCP-1 (25542-1-AP),COX-2 (12282), p65 (8242), p-p65 (3033), Mfn1 (13798-1-AP), Mfn2 (12186-1-AP),HO-1 (A19062), Drp1 (12957-1-AP), GCLC (12601-1-AP), Opa1 (27733-1-AP),GCLM (14241-1-AP), Fis1 (10956-1-AP), Nrf2 (16396-1-AP), and Keap1(10503-2-AP) were purchased from Cell Signaling Technology , Abclonal , Proteintech , Abcam, and Affinity . Recombinanthuman and mouse sEH afforded from Prof. Bruce D. Hammock  were gifts.WED was isolated and identifiedfrom 2 at 37 °C. Overnight, cells were treated with WED. After theincubation for 24 h, the cells were collected for the cellular viabilityusing the CCK-8 kit. The cells were seeded into the 96-well plateovernight and pretreated with WED  for 1 hbefore the challenge with LPS (500 ng/mL). After 24 h, the supernatantswere collected and analyzed for the anti-inflammatory and antioxidanteffects of WED.RAW264.7 macrophages wereseeded into 96-well plates and cultured in DMEM with 10% fetal bovineserum (FBS) at 37 °C in humidified air containing 5% COFor the sEH rescue experiment, cells were pretreatedwith sEH  for 1 h, and incubated with WED (20μM). After 24 h of LPS exposure, the cells were harvested forPCR and Western blot analyses.For the inhibition experimentof GSK3β, cells were pretreatedwith the GSK3β inhibitor LiCl (5 mM) or 14,15-EET (5 μM)for 1 h before WED (20 μM) treatment. Then cells were administratedLPS for 24 h before being harvested for PCR and Western blot analyses.Ephx2 (siEphx2) and negative control siRNA(siCtrl) were designed and synthesized by GenePharma .siCtrl (5 μL) and siEphx2 (5 μL) weretransiently transfected into RAW264.7 cells using the transfectionreagent (5 μL). After 6 h of transfection, fresh DMEM was addedwhen replacing the medium, and the cells were continuously incubatedfor 36 h. The cells were harvested for both PCR and Western blot analysesto evaluate the efficiency of siEphx2 silencing.For the confirmatory experiment of targeting sEH with WED, wild-type(WT) and siEphx2 cells were pretreated with WED for1 h before the LPS challenge (500 ng/mL). Cells were harvested forPCR and Western blot analyses after 24 h of incubation.Small interfering RNA of Cells were pretreated with WED for 1 h before the challenge with LPS. After 24 h, the ROS were measuredby using probe DCFH-DA for 30 min at 37 °C. The cells were collectedand used for the analysis of ROS-positive cells through the flow cytometryand a Leica DM4B microscope .44 Cell lysates were incubated with WED beads orWED overnight at 4 °C, and PBS was used to elute the nonspecificbinding proteins three times. The bead-captured proteins were analyzedby silver staining, Western blot, and a LC-MS/MS system.Epoxy-activated Sepharose6B beads  coupled with WED were conductedaccording to the manufacturer’s protocol, as previously described.Figures S19–S22), as previouslydescribed,53 for the colocalization ofWED and sEH.WEDcoupled with biotin (Bio-WED) was synthesized and identified by NMR(Cell lysates wereincubated with WED (50 μM) for 30 min at 4 °C. The variabletemperature experiments were performed at 46, 50, 54, 58, and 62 °Cfor 3 min, and the supernatants, afforded by the centrifugation, wereanalyzed by Western blot using the sEH antibody. The vehicle was usedas the control group.Cell lysates pretreated with WED  for 1 hwere treated with Pronase (7.5 μg/mL) and incubated for 15 min.After the centrifugation, the supernatants were analyzed by Westernblot using the sEH antibody. The vehicle was used as the control group.Kd) of WED with sEH was fitted as previously reported.54WED (0.1 μM)was incubated with different concentrations of human recombinant sEHin PBS for 2 min, and then, the fluorescence signal was recorded ona microplate reader. The dissociation constant (55 different concentrations of WED were added in the standard bufferwith the labeled sEH protein (200 nM) for the incubation (15 min)and then analyzed on a Monolith NT.115 instrument  at room temperature. All the data were analyzedby the NT analysis software to afford the Kd value of WED with sEH.First, usingthe Monolith NT kit to label the sEH protein,Ephx2+/+) and sEH KO (Ephx2–/–) C57BL/6 mice  wereobtained from the Experimental Animal Center of Dalian Medical University and Cyagen Biosciences Inc. , respectively,and kept under 12 h of light and 12 h of dark environment with a controlledtemperature (22–24 °C) and humidity (50–60%).WT  and LPS (5 mg/kg) were dissolvedin 10% hydroxypropyl β-cyclodextrin and saline, respectively,and then stored at 4 °C. Mice were randomly classified into sixgroups, including the control, WED (20 mg/kg), LPS, LPS + WED (5 mg/kg),LPS + WED (10 mg/kg), and LPS + WED (20 mg/kg) groups. WED  was administrated intragastrically to mice in the WEDand LPS + WED groups for a week by intragastric infusion, followedby administration of LPS (50 μL) through the intratracheal instillationafter 1 h of the last administration of WED. Mice in the control andLPS groups were conducted with the corresponding vehicle or LPS (50μL), according to the abovementioned protocol. After 24 h ofLPS administration, BALF was collected as follows: the lungs werelavaged with 0.5 mL saline thrice, and the recovered fluid was usedto analyze the levels of proteins IL-6 and TNF-α; activitiesof MPO and LDH; and the number of WBCs, PMNs, and MNs.Ephx2+/+ and Ephx2–/– mice were classified into four groups(10 mice/group): the LPS (5 mg/kg)-treated Ephx2+/+ group, the LPS (5 mg/kg) + WED (20 mg/kg)-treated Ephx2+/+ group, the LPS (5 mg/kg)-treated Ephx2–/– group, and the LPS (5mg/kg) + WED (20 mg/kg)-treated Ephx2–/– group. Ephx2+/+ and Ephx2–/– mice were evaluated using the abovementionedprotocol, and the lungs were collected.Second, After being fixedwith 4% paraformaldehyde for 24 h and embedded with paraffin, thelungs were sliced into 4 μm thick sections, which were thenused for the pulmonary pathological analysis according to the Hematoxylinand Eosin  kit.g) for 20 min at 4 °C, and then, levels of 8,9-EET,11,12-EET, 14,15-EET, 8,9-DHET, 11,12-DHET, and 14,15-DHET were calculatedon the basis of their standard curves and peak areas afforded froman AB Sciex Qtrap 5500 LC-MS/MS system , respectively,as previously reported.24The supernatant of the lung samplewas collected after homogenization and centrifugation  challenge. After fixation,the cells were successively incubated with primary antibodies p65,Keap1, or Nrf2 at 4 °C overnight and the fluorescent secondaryantibody for 1 h and then analyzed with a Leica DM4B microscope .For the colocalization of WED with sEH, the cellswere incubated with Bio-WED or the vehicle in 6-well plates for 1h. After fixation, the cells were incubated with primary sEH antibodyat 4 °C overnight and the fluorescent secondary antibody for1 h and then analyzed by a Leica DM4B microscope .For the lung samples, 10% goat serum was used for blocking microwavedlung sections for 20 min, followed by incubation with a cluster ofCD68, Gr-1, TNF-α, HO-1, and Nrf2 antibodies overnight at 4°C. The sections were washed with PBS three times before theincubation with normal or fluorescent secondary antibodies. Finally,the sections were used for the immunohistochemical and immunofluorescentanalyses.The concentrations of protein in cell, BALF, and lung samples weremeasured using the BCA method. The activities of MPO, LDH, and SODand the contents of MDA and GSH in BALF and lung were measured usingthe appropriate kits . The levels of TNF-α and IL-6 were determined using theircorresponding ELISA kits .COX-2, Mfn1, Mfn2, Opa1, Drp1, MCP-1, ICAM-1, Fis1, HO-1, TNF-α, NQO-1, GCLC, IL-6, GCLM, Keap1, iNOS, and Nrf2 were added in the cDNA afforded after the reverse transcription,together with TransStart Tip Green qPCR SuperMix , respectively, and then real-time qPCR was performed on anApplied Biosystem 7500 Real-time PCR System . The copy number of every gene was normalized to the referencegene β-actin, and its relative mRNA expression was determinedon the basis of the 2–ΔΔCt method.The total RNA in the cellsand lungs was extracted with a TRIzol reagent, and its quantity andpurity were analyzed in a Nanodrop spectrophotometer . The primers of –ΔΔCt method.For the copy number of mtDNA, the DNA was determined using the UniversalGenomic DNA Purification Mini Spin Kit and assayed using the primers of mtDNA by real-time qPCR on the basisof the 2Cell and lung samples were added in thelysis buffer with cocktail, homogenized, and centrifuged  to obtain total proteins. Protein (20–30μg) was loaded on 8%–12% SDS-PAGE for electrophoresis,and the polyvinylidene difluoride (PVDF) membranes transferred withthe target protein were incubated with the corresponding primary antibodiesovernight at 4 °C after blocking with 5% skim milk for 2 h. Afterthe incubation with a horseradish peroxidase-conjugated secondaryantibody, the membranes were incubated with the ECL reagent, followedby detection on the Tanon 5200-ECL detection system.Cell lysates were treatedwith WED (20 μM) at 4 °C for 30 min and then incubatedwith sEH or IgG antibody and protein A/G magnetic beads at 4 °Covernight. The resulting protein was analyzed by Western blot andthe LC-MS/MS system.57The inhibitionof human and mouse sEH by WED was evaluated using PHOME as the substrate,as previously described.57 and plotted by PyMOL 2.4 software.The interaction of WEDwith sEH (PDB: 4OCZ) was analyzed using the GROMACS package, accordingto previous methods,p-value was less than 0.05, the result was considered significant.All the data are presented asmeans ± standard error of the mean (SEM) and analyzed on thebasis of one-way ANOVA followed by Tukey’s test in Prism GraphPadPrism 8.0, except for the data in the experiments of sEH knockdown,sEH rescue, the inhibition of GSK3β by LiCl, and sEH KO experiment(two-way ANOVA followed by Sidak’s test). If the


Dopamine regulates several functions, such as voluntary movements, spatial memory, motivation, sleep, arousal, feeding, immune function, maternal behaviors, and lactation. Less clear is the role of dopamine in the pathophysiology of type 2 diabetes mellitus (T2D) and chronic complications and conditions frequently associated with it. This review summarizes recent evidence on the role of dopamine in regulating insular metabolism and activity, the pathophysiology of traditional chronic complications associated with T2D, the pathophysiological interconnection between T2D and chronic neurological and psychiatric disorders characterized by impaired dopamine activity/metabolism, and therapeutic implications. Reinforcing dopamine signaling is therapeutic in T2D, especially in patients with dopamine-related disorders, such as Parkinson’s and Huntington’s diseases, addictions, and attention-deficit/hyperactivity disorder. On the other hand, although specific trials are probably needed, certain medications approved for T2D  may have a therapeutic role in such dopamine-related disorders due to anti-inflammatory and anti-oxidative effects, improvement in insulin signaling, neuroinflammation, mitochondrial dysfunction, autophagy, and apoptosis, restoration of striatal dopamine synthesis, and modulation of dopamine signaling associated with reward and hedonic eating. Last, targeting dopamine metabolism could have the potential for diagnostic and therapeutic purposes in chronic diabetes-related complications, such as diabetic retinopathy. Dopamine has several systemic effects and may have a role in the pathophysiology of chronic diseases. This review aims to summarize the leading evidence regarding the pathophysiological and potentially therapeutic role of dopamine in type 2 diabetes (T2D) and to explore the cross-linking between T2D pathophysiology and the most frequent chronic neurologic and psychiatric disorders associated with impaired dopamine activity/metabolism and T2D in human pathology.8H11NO2) is a catecholamine derived by the amino acid tyrosine after a two-step reaction involving (a) the enzyme tyrosine hydroxylase (step 1), which transforms the amino acid tyrosine in L-dihydroxyphenylalanine (L-DOPA), and (b) the enzyme DOPA decarboxylase (step 2), which converts L-DOPA in dopamine [Dopamine C8H1NO2 is aDopamine is synthesized mainly in the brain and other tissues with neuroectodermal origin, such as the medulla of the adrenal glands and paraganglia, where it serves as the precursor of adrenaline and noradrenaline synthesis. Dopamine is well known as a neurotransmitter, but extracerebral sites of production and dopamine receptors (DRs) are also widely expressed in peripheral tissues.2 receptor antagonists), resulting in irregular muscle contractions manifesting as tremors, spasms, motor restlessness, and tardive dyskinesia. In the tuberoinfundibular pathway, dopamine neurons begin in the hypothalamic arcuate and periventricular nuclei and project to the median eminence of the hypothalamus. After that, dopamine is released into the portal circulation, connecting the median eminence to the pituitary gland, where dopamine inhibits the prolactin release from lactotrophic cells. A mild or moderate increase in serum prolactin is a secondary effect of D2 antagonists [Four dopaminergic pathways exist in the central nervous system: the mesolimbic, mesocortical, nigrostriatal, and tuberoinfundibular . In the agonists .3 stimulation, as demonstrated by experiments using specific dopamine antagonists [2 and DR3 signaling [The potential role of endogenous catecholamines in the pathogenesis of T2D was suggested by landmark studies in the 1970s ,7,8. Theagonists . In anotagonists . Apart fagonists . An inveagonists , in whicignaling ,19. In aignaling . Glucoseignaling . As an aignaling .Given the anti-secretive and antiproliferative effects, dopamine may have a role in the pathophysiology of T2D. Monoaminoxidase A and B play a crucial role in the catabolism of catecholamines, including dopamine. Both isoforms are also expressed in β-cells, and a lower level of monoaminoxidase activity is associated with dampened insulin secretion. Therefore, this evidence suggests that blunted dopamine catabolism and, consequently, high intra-islet dopamine concentration may contribute to reducing insulin secretion and raising the number of apoptotic β-cells, both events primarily involved in the pathophysiology of T2D. Interestingly, the transcription of monoaminoxidase A and B genes is under the MAF transcription factor A control . MAF traThese data suggest that dopamine and insulin may be involved in a potential feedback mechanism in which one negatively regulates the metabolism of the other .Pivotal studies suggested that intravenous dopamine infusion stimulated glucagon release  in a dosT2D and chronic comorbidities, such as arterial hypertension, overweight/obesity, and dyslipidemia, foster the development of chronic complications over time . HyperglThe role of dopamine in the pathophysiology of diabetes-related chronic complications is an emerging issue . Compreh1 and DR2 are the most widely expressed receptors mediating dopamine activity in the whole body [Dopamine and DRs in the nephron tubules are essential in regulating key renal functions, such as electrolytes and water resorption, acid–base balance, and blood pressure regulation. DRole body . In chroole body . Becauseole body . Experimole body . One pilole body .2 agonists may improve hemodynamics in T2D patients with heart failure (HF), positively affecting heart-failure-related outcomes [Dopamine plays many actions in the human heart, including positive inotropic and chronotropic effects, regulation of coronary flow, and cardiomyocyte metabolism . These eoutcomes . Neverthoutcomes . Especiaoutcomes ,67. Antaoutcomes . From a outcomes . Neverthoutcomes ,71. It iNeurologic effects after ischemic stroke largely depend on the location and extension of ischemic areas, time of exposure to ischemic reperfusion injury, and baseline cerebral performance. Generally, ischemic stroke impairs dopamine release, synthesis, and DR activity in the striatum . DopaminDopamine plays a crucial role in the central nervous system (CNS), as it regulates many activities, including motor control, spatial memory, motivation, sleep, arousal, feeding, immune function, maternal behaviors, and lactation, to cite the main effects . DopaminPD is characterized by a primitive degeneration of a group of neurons in the substantia nigra and largely synapsing with basal ganglia. The distinctive neuropathological hallmarks are the Lewy bodies, α-synuclein aggregates, located in the substantia nigra with overtime spreading to neocortical and cortical regions (late-stage disease) . DopaminHuntington’s disease (HD) is a neurodegenerative autosomal dominantly inherited disorder caused by a mutation of the Huntingtin gene located in chromosome 4. The mutation consists of a progressive  trinucleotide (CAG) triplet expansion, resulting in an extended polyglutamine sequence into the Huntingtin protein. When the number of CAG triplet repetitions is more than 35, the mutation makes missense changes in the native Huntingtin structure, compromising its functions, and the disease is most likely to occur . Hunting4 and DR5 [2 agonism, which is usually impaired in ADHD as well as obesity, by GLP-1RAs would result in caloric restriction and weight loss [Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder defined by impairing levels of inattention, disorganization, and/or hyperactivity–impulsivity . The est and DR5 . Real-wo and DR5 , and it  and DR5 . The res and DR5 . In addi and DR5 . Risks s and DR5 . The imp and DR5 . Compuls and DR5 ,169, and and DR5 . Targeti and DR5  with pos and DR5 , and bod and DR5 ,174. Nev and DR5 . So far, and DR5 . These aght loss ,178. Bes2 and reduced neuronal metabolic activity in these areas, leading to an impaired reward system [Addiction refers to a psychopathological condition in which one individual is unable to control a specific impulse to carry out reiterating actions or behaviors that hesitate in physical or psychological dependence on something . In phard system . Conversd system . Low andd system ,198. Desd system . No specd system ,201, so d system . The caud system ,204,205.d system . The VTAd system . After td system . Most imd system . Ghrelind system ,210. Thid system  and neurd system . On the d system . Interesd system . Similard system . Despited system ,217. In d system ,219,220.Here, we discuss the pharmacological role of dopamine in T2D and of anti-hyperglycemic medications in T2D and concomitant diseases in which dopamine activity/metabolism is impaired to overview existing and lacking evidence indicating that specific drugs may improve the pathophysiology and clinical manifestation of the above-mentioned disturbances. Most evidence refers to PD due to its more epidemiological relevance than other dopamine-related conditions. However, more information about the potential effects of approved treatments for T2D and chronic neurologic and psychiatric disorders characterized by impaired dopamine activity/metabolism on specific disease-related outcomes has been summarized in 2 receptor signaling [2 overexpression abolished the glucose-stimulated Ca2+ influx and insulin secretion in β-cells. This toxic effect was partially reverted after the treatment with a D1-D2 heterodimer agonist (D1 and D2 dimerized on β-cell surface), suggesting that the D1 receptor may protect β-cells from the harmful effects of dopamine by modulating D2 signaling [2 receptors in mediating the intrapancreatic effects of dopamine [The intrapancreatic uptake of levodopa increases after glucose exposure, and hyperglycemia may sustain the machinery of dopamine synthesis in β-cells. Animal models showed that dopamine directly suppresses insulin secretion by curbing the duration of the action potential in response to β-cell glucose entry. The leading effect is mediated by the Dignaling . In an eignaling . Given tdopamine , specifidopamine ,225.2 agonist, is an FDA-approved oral medication for T2D, available in the US as tablets of 0.8 mg. The maximal approved daily dose is 4.8 mg, corresponding to 8 tables per day [2 on β- and α-cells, respectively [Bromocriptine, a DR per day . The tab per day . Bromocr per day . Thanks ectively . Bromocrectively . Dopaminectively . Dopaminectively . The benectively ,233. Howectively ,235. Oth2 signaling is increased in islets from high-fat-diet obese mice. The whole grain-derived γ-oryzanol, a mixture of vegetal sterols, improved glucose control, promoted weight loss, and reduced appetite and caloric intake by acting at the hypothalamic level [An experimental study indicated that the expression of molecules involved in DRic level . Oryzanoic level . With a ic level . Moreoveic level . Preclinic level ,241. OveMetformin is a synthetic biguanide approved as first-line treatment in T2D due to several effects, including mild suppression of gluconeogenesis in the liver, reduction in glucose absorption in the gut, enhancement of glucose utilization by mitochondria in peripheral tissues, and induction of weight loss . Most ofAcarbose, an α-glucosidase inhibitor, attenuates the intestinal absorption of carbohydrates. Thanks to this mechanism, acarbose improves glucose control, especially by reducing post-prandial glucose amplitude and insulin spikes . In addiApart from the anti-hyperglycemic properties, peroxisome-proliferator-activated receptor (PPAR) agonists have shown promising neuroprotective effects. In T2D, the foremost PPAR-γ agonist, pioglitazone, has relevant anti-inflammatory and anti-atherosclerotic properties . SeveralDipeptidyl peptidase IV inhibitors (DPP-IVis) are a class of oral gliptins approved for T2D. These agents compete with endogenous incretins at the catalytic site of the enzyme and delay the incretin degradation . TherefoGLP-1RAs are promising agents. As mentioned above, Exenatide provided reliable evidence of improving motor and non-motor symptoms in PD, but its role in reducing the pathophysiology and background brain injury in humans currently needs to be clarified . PromisiMore recently, the glucose-dependent insulinotropic peptide (GIP) has gained endorsement as a promising therapy in T2D. Dual agonists GIP/GLP-1RA, rather than single GIP agonists, have been shown to improve glucose control and induce relevant weight loss significantly better than previously observed with GLP-1RAs alone ,297,298.SGLT2is impairs glucose resorption at the level of the proximal tubule in the kidney . Thanks Dopamine signaling is involved in the fine regulation of several physiologic mechanisms that control cerebral functions, cognition, eating behaviors and reward, maintenance of glucose balance, and retinal, renal, and cardiovascular homeostasis. Impaired dopamine synthesis, metabolism, or activity is associated with neurological and psychiatric diseases, impaired glucose metabolism and T2D, and diabetes-related chronic complications. Evidence suggests that reinforcing dopamine signaling has a therapeutic role in T2D. The therapeutic implication should be better investigated in patients with dopamine-related disorders, such as PD, HD, addictions, and ADHD, as they are exposed to an increased risk of T2D, indicating the existence of a cross-link among these conditions. Understanding the potential interaction between pharmacological interventions in such disorders may ameliorate the management of patients, but specific trials are needed to confirm the therapeutic potential of certain medications. Last, emerging evidence suggests that dopamine imbalance is involved in developing chronic diabetes-related complications, and targeting dopamine metabolism would have the rationale for diagnostic and therapeutic purposes, as found in diabetic retinopathy.