Inhibited immune response and low levels of delivery restrict starvation cancer therapy efficacy. Here, we report on the co-delivery of glucose oxidase (GOx) and indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan using a metal-organic framework (MOF)-based nanoreactor, showing an amplified release for tumor starvation/oxidation immunotherapy. The nanosystem significantly overcomes the biobarriers associated with tumor penetration and improves the cargo bioavailability owing to the weakly acidic tumor microenvironment-activated charge reversal and size reduction strategy. The nanosystem rapidly disassembles and releases cargoes in response to the intracellular reactive oxygen species (ROS). GOx competitively consumes glucose and generates ROS, further inducing the self-amplifiable MOF disassembly and drug release. The starvation/oxidation combined IDO-blockade immunotherapy not only strengthens the immune response and stimulates the immune memory through the GOx-activated tumor starvation and recruitment of effector T cells, but also effectively relieves the immune tolerance by IDO blocking, remarkably inhibiting the tumor growth and metastasis in vivo. Inhibited immune response and low levels of delivery inhibit starvation cancer therapies. Here, the authors report on the co-delivery of glucose oxidase and IDO inhibitor 1-methyltryptophan using metal organic frameworks and show amplified release in response to starvation therapy along with immune modulatory effects. GOx has been applied for cancer starvation therapy, due to its ability to exhibit the immunostimulatory effects4. It can effectively kill tumors by emulatively depleting glucose and generating cytotoxic reactive oxygen species (ROS), thus, facilitating the exposure of tumor-associated antigens (TAAs) for an overall antitumor effect5. However, its immunostimulatory effect is naturally inhibited owing to various negative feedback immune resistance mechanisms6. The blockade of negative regulatory pathways combined with starvation/oxidation therapy represents one of the most promising strategies for tumor immuotherapy7.The starvation therapy represented by glucose oxidase (GOx) has been recognized as a “green” strategy against cancer, as it cuts off the necessary nutrient supply to tumors with negligible side effects9. The recent studies suggested that the IDO-specific competitive inhibitor, i.e., 1-methyltryptophan (1-MT), could effectively relieve immune evasion12. However, a modest anticancer immunity was demonstrated for the IDO-blockade monotherapy due to insufficient antigen presentation and immune response13. Thus, the combination of the 1-MT-mediated IDO blockade immunotherapy and GOx-activated starvation/oxidation can be a feasible strategy against tumors with strong immune response and weak immune resistance.The immune checkpoint protein indoleamine 2,3-dioxygenase (IDO) is highly expressed in tumors, which can inhibit the effector T cell proliferation and induce the expansion of the T regulatory (Treg) cells by catalyzing tryptophan (Trp) to kynurenine (Kyn), thereby presenting itself as an attractive immune-therapeutic target for relieving immunosuppressive microenvironment15. On one hand, the metal-organic framework (MOF)-based nanoreactors combining the advantages of MOFs  and nanoreactors  have been proposed as ideal vehicles17, owing to efficient codelivery of the non-toxic biological enzymes  and 1-MT to tumors. Thus, the in situ cargo release and substrate catalysis  lead to the generation of toxic species  with improved bioavailability and therapy efficacy. On the other hand, the tumor microenvironment-activated size/charge changeable strategy can overcome these biological barriers, leading to the improved delivery efficiency and therapeutic effect19. Therefore, the MOF-based nanoreactors with the size/charge changeable features represent an appropriate nanosystem for boosting the antitumor immune response through starvation/oxidation combined IDO-blockade immunotherapy.As the poor bioavailability and rapid inactivation of GOx and sequential biological barriers result in limited tumor penetration and low endocytosis, the construction of multifunctional nanosystems for efficient transfer of GOx and 1-MT in tumors is crucial to enhance the therapy efficacy2+, which can be rapidly disassembled triggered by the rich intracellular ROS of tumor cells, thus, minimizing the long-term retention toxicity of the conventional MOFs; (2) The size/charge changeable strategy designed in the nanoreactor sequentially breaks the biobarriers and improves the delivery efficiency. The shielding shell of the PCP-Mn-DTA@GOx@1-MT nanosystem exhibits a rapid removal of polyethylene glycol (PEG) component to afford a polyethylenimine (PEI)-conjugated cationic core in response to the weakly acidic tumor microenvironment (pH~6.8). The transformed nanosystem with strongly positive charge and small size significantly improves the tumor penetration depth and endocytosis; (3) The consumption of glucose by GOx is accompanied by the elevated generation of H2O2, which can be further converted to hydroxyl radical (·OH) with high toxicity through Mn2+-mediated Fenton-like reaction, thus leading to the complete MOF degradation, drug release and improved therapeutic efficacy; (4) Taking advantage of the promoted immune response by GOx-mediated starvation/oxidation therapy and immune resistance suppression executed by IDO blockade immunotherapy, the PCP-Mn-DTA@GOx@1-MT nanosystem presents a remarkable therapeutic effect. Therefore, the successful construction of the multifunctional PCP-Mn-DTA@GOx@1-MT nanoreactor provides a paradigm for effectively overcoming the delivery biobarriers and revealing a superior tumor killing efficacy through starvation therapy along with immune modulatory effects.In this work, a pH/ROS dual-sensitive degradable MOF nanoreactor-based nanosystem (denoted as PCP-Mn-DTA@GOx@1-MT) with self-amplified drug release and enhanced tumor penetration has been constructed to co-deliver GOx and 1-MT for the tumor starvation/oxidation/IDO-blockade immunotherapy. Compared to previous studies, the present nanosystem exhibits following four important advantages  was synthesized , which was confirmed by 1H NMR and mass spectrometry , a ROS-sensitive compound try Fig. . Mn-DTA try Fig. , consisture Fig. . In partgon Fig. . These r23. The structure of Mn-DTA was further verified by energy-dispersive X-ray spectroscopy (EDS) elemental mappings of Mn, S and O . As shown in Fig. d O Fig. , origina3 as the shell on the surface of the Mn-DTA core. Compound 3 was successfully synthesized as reported previously19 and confirmed by 1H NMR spectroscopy, gel-permeation chromatography (GPC), and Fourier-transform infrared (FTIR) spectroscopy , as confirmed by dynamic light scattering , the average size of PCP-Mn-DTA@GOx@1-MT dramatically decreased to around 55 nm , due to the self-generation of ROS and cascade-amplified drug release triggered by GOx. It was worth noting that PCP-Mn-DTA@GOx@1-MT exhibited a sufficient drug release when exposed to the biologically relevant glucose and intratumoral ROS concentration  was observed in the control group under physiological conditions (pH 7.4) for 24 h Fig. , suggest26. Typically, free GOx and PCP-Mn-DTA@GOx@1-MT were incubated with glucose to evaluate the GOx activity, which was reflected by the pH change (induced by gluconic acid) and H2O2 generation. As shown in Fig. 2O2 generation increased, along with the catalytic reaction duration, in both GOx and PCP-Mn-DTA@GOx@1-MT groups. The reduction of pH and generation of H2O2 in PCP-Mn-DTA@GOx@1-MT rapidly reached equilibrium within 1 h, and a similar trend was also observed in free GOx. These results confirmed a high catalytic activity of the PCP-Mn-DTA@GOx@1-MT nanosystem, which was not affected by the MOF encapsulation. Compared with the control and PCP-Mn-DTA negative groups, both PCP-Mn-DTA@GOx and PCP-Mn-DTA@GOx@1-MT groups effectively generated ROS, as revealed by the green fluorescence and related quantitative analysis . The results confirmed that PCP-Mn-DTA@GOx@1-MT nanosystem could achieve an efficient drug release, and ROS generated by the nanosystem was indeed glucose-dependent.To reveal the catalytic activity of GOx and ROS self-generation capacity of the nanosystem, the enzyme activity test and ROS probe-based confocal laser scanning microscopy (CLSM) were employedsis Fig. . The amo−1) regardless of the incubation time , and CLSM was used to monitor the penetration of the nanosystem. As shown in Fig. The B16F10 cell-based multicellular spheroids (MCSs) were constructed for evaluating the tumor infiltration property of the nanosystem31.The cellular uptake level of the nanosystem in the B16F10 cells was subsequently investigated by flow cytometry (FCM) and CLSM. As compared to the control, the uptake of PCP-Mn-DTA@GOx@1-MT by the B16F10 cells exhibited a time-dependent behavior, and the endocytosis amount of PCP-Mn-DTA@GOx@1-MT was remarkably higher than free GOx at pH 7.4 or pH 6.8 Fig. , indicat32. Furthermore, both PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT inhibited the downregulation of IDO expression more significantly as compared to free 1-MT  and PCP-Mn-DTA negative groups, 1-MT displayed an obvious IDO expression inhibition Fig. 32. Furth33. As shown in Fig. + T cells was significantly increased after treatment with 1-MT and 1-MT loaded formulations (PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT), thus, implying the improved T cell proliferation. Moreover, the amount of the proliferated T cells in PCP-Mn-DTA@GOx@1-MT was larger than the 1-MT group  and red florescence (lysotracker red marked lysosomes)ane Fig. , demonstvs. 21.7%, p < 0.01, Fig. 37 and the delivery effect of the nanosystem. PCP-Mn-DTA@GOx@1-MT led to a severe apoptosis (47.4%), resulting from the GOx-generated starvation/oxidation damage. Obvious H2O2 generation and inhibition of the ATP production by the GOx-loaded systems  and live-dead staining kits. As shown in Fig. To further illustrate the apoptosis mechanism induced by the PCP-Mn-DTA@GOx@1-MT nanosystem, the western blot assay was employed to evaluate the expression of the related apoptosis proteins, including Bax, Bcl-2 and Cyt-C. As demonstrated in Fig. 39, the degree of anti-migration of PCP-Mn-DTA@GOx@1-MT was evaluated by the wound healing, tumor migration and invasion studies. As shown in Fig. p < 0.01, Fig. As the energy metabolism regulated by GOx and 1-MT was closely associated with the tumor migration41 as well as high drug delivery efficiency. Remarkably, PCP-Mn-DTA@GOx@1-MT induced the highest extent of tumor-infiltrating CTLs and lowest amount of Treg cells regardless of the B16F10 and 4T1 tumor-bearing mouse models  and activation of the signal transducers/activators of transcription (STAT3) through the AHR-IL-6-STAT3 pathway46, the proliferation of the matured dendritic cells was investigated to further illustrate the mechanism of the nanosystem for boosting the immune response. As demonstrated in Fig. p < 0.01, Fig. As the effective tumor cell killing is positively associated with the level of the matured dendritic cells as well as the reinforcement of the immune response activation by exposing TAAs and presenting the “eat me” signalTo authenticate the advantage of the combined starvation/oxidation therapy and IDO-blockade immunotherapy, the antitumor evaluation was simultaneously performed in vivo in both B16F10 and 4T1 tumor-bearing mouse models. In these models, the PCP-Mn-DTA group exhibited a rapid tumor growth similar to the control Fig. ’, indica47, leading to effective recruitment of the effector T cells. Furthermore, 1-MT from PCP-Mn-DTA@GOx@1-MT could significantly suppress the IDO expression and relieve IDO-mediated immune tolerance, resulting in the recruitment of CTLs, B cells and NK cells as well as the inhibition of Treg cell proliferation. Lastly, the PCP-Mn-DTA@GOx@1-MT nanosystem combined with the starvation/oxidation therapy and IDO-blockade immunotherapy provided the most significant antitumor effect with reinforced immune activation and weakened immune tolerance.The excellent antitumor and anti-metastasis effects of the PCP-Mn-DTA@GOx@1-MT nanosystem could be explained as follows. Firstly, the nanosystem endowed with the pH-responsive size/charge transformation could markedly increase the tumor permeability and cellular uptake, thereby improving the delivery efficacy and bioavailability of GOx and 1-MT. Secondly, GOx from PCP-Mn-DTA@GOx@1-MT could effectively kill the tumor cells and expose TAAs via starvation/oxidationSubsequently, H&E, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and immunofluorescence (IFC) staining analysis were conducted to further prove the comprehensive antitumor activity of the nanosystem in vivo. PCP-Mn-DTA@GOx@1-MT induced the most severe tumor damage, as revealed by the distinct tissue dissociation in the H&E images and numerous magenta dots in the TUNEL observation Fig. ’. In add2O2 production after intravenous injection of PCP-Mn-DTA@GOx@1-MT. The photoacoustic images and quantitative results indicated that the sO2 average displayed a ~81% drop after the administration of PCP-Mn-DTA@GOx@1-MT for 24 h, and the photoacoustic imaging intensity from the tumor site was correspondingly enhanced  the GOx-induced generation of H2O2 further amplified the disassembly of the nanosystem and GOx release through a cascade reaction; (3) the ·OH radical generated from the Fenton-like reaction between Mn2+ and H2O2 contributed to the augmented photoacoustic signal as well48. Notably, the quantitative analysis of melanin in mice demonstrated that PCP-Mn-DTA@GOx@1-MT significantly reduced the amount of intratumoral melanin, while there was an insignificant change in the GOx negative group  and Hced Fig. . In compoup Fig. . These r49. Thirdly, no obvious organ damage and body weight loss was observed in the case of PCP-Mn-DTA@GOx@1-MT group in both B16F10 and 4T1 tumor models, as revealed by the histological and H&E analysis , attributed to the shielding effect and negative charge endowed by the PEG outer layer50, as well as the reduced size and charge reversal design of the nanosystem.Furthermore, the biological safety of the nanosystem was studied. Firstly, the PCP-Mn-DTA@GOx@1-MT did not induce a continuous decrease in the peripheral blood glucose as compared to the control Fig. , suggestEM) at day 8  correspondingly decreased (p < 0.01). It confirmed the long term secondary immune response effect. The results indicated that the PCP-Mn-DTA@GOx@1-MT nanosystem could successfully initiate the immune memory response with a long-lasting effectiveness. Thus, the multifunctional PCP-Mn-DTA@GOx@1-MT nanosystem with the size reduction and charge conversion abilities could synergistically kill the tumor cells and suppress the tumor metastasis via combined advantages of starvation/oxidation-reinforced immune response initiation and IDO-blockade immune tolerance suppression.Lastly, the tumor re-challenge and secondary response of the immune-memory T cell assays were conducted to systematically evaluate the persistence of the protective immune effect and potential clinic application of the nanosystem Fig. . As show 8 Figs. ’ and 50  8 Figs.  under thIn this study, a pH/ROS dual-responsive degradable MOF nanoreactor-based nanosystem has been developed with self-amplified release and enhanced penetration to co-deliver GOx and 1-MT for the combined starvation/oxidation therapy and IDO-blockade immunotherapy of tumors. The detailed in vitro and in vivo results have authenticated that the PCP-Mn-DTA@GOx@1-MT nanosystem not only strategically overcomes the biobarriers and improves the delivery efficiency via the weakly acidic tumor microenvironment-sensitive size/charge transition, but also effectively reinforces the immune response activation with reduced immune tolerance via the GOx-activated starvation/oxidation therapy and IDO-blockade immunotherapy. By taking advantage of these strategies, the PCP-Mn-DTA@GOx@1-MT nanosystem effectively suppresses the tumor growth and metastasis. Therefore, this study provides a promising paradigm for overcoming the biobarriers and reinforcing the immune response activation as well as relieving the immune resistance by the starvation/oxidation-integrated IDO-blockade immunotherapy.1 (yield 81%). 1H NMR : 2.85 , 2.58 , 1.58 . ESI-MS: Calc. 251.0417, found 251.0404. The 1H NMR and mass spectra were presented in Fig. 3-Mercaptopropionic acid (4.9 mmol) was dissolved in acetone (9.82 mmol) and stirred for 8 h. Subsequently, the solution was crystallized in an ice bath overnight. The filtrated crystals were repeatedly washed by cold water and hexane, followed by drying under vacuum to obtain compound 2 solution , compound 1 , polyvinylpyrrolidone  and triethylamine were added to a centrifuge tube. Afterwards, the DMF/ethanol mixture was added until the volume was 13 mL. Subsequently, the solution was transferred to a reactor and held at 150 °C for 24 h. Finally, the product, termed as Mn-DTA, was collected by centrifugation  and was re-dispersed in ethanol for further use.The MnCl2 (PEG-CDM) was harvested via vacuum drying. 1H NMR : 7.95 , 3.71 , 3.45  and 2.93 . FTIR: 2885, 1726, 1633, 1569, 1471 and 1115 cm−1. The 1H NMR and FTIR spectra of 2 were shown in Supplementary Fig. Briefly, Cis-aconitic anhydride  and oxalyl chloride (2 equiv.) were dissolved into dry dichloromethane at 0 °C. Next, DMF was added dropwise and, the mixture was transferred to the room temperature condition for 2 h. The chloride acetylated CDM was harvested via vacuum drying and then reacted with mPEG-OH in the presence of pyridine for another 2 h. The saturated ammonium chloride was then added to terminate the reaction. The organic phase was extracted and precipitated by ice-cold ether twice. Compound 2 (0.6 mmol) were mixed with dimethyl sulfoxide  at 0 °C. Afterwards, 4-dimethylaminopyridine (1.2 mmol) was added dropwise and stirred for 0.5 h. The reaction contents were warmed to room temperature in the dark, and the reaction was continued for 2 h. The mixture was then dialyzed (MWCO 3.5 kDa) against double distilled water for 3 days. Compound 3, also denoted as PEG-CDM-PEI, was achieved by the lyophilization (yield: 82%). 1H NMR : 3.83 , 3.31  and 2.62-2.97 . The molecular weight was determined to be 7200 by GPC, with a polydispersity index of 1.29. FTIR: 2885, 1739, 1645, 1562, 1471 and 1116 cm−1. The 1H NMR, GPC and FTIR spectra of 3 were shown in Supplementary Fig. Branched PEI (0.9 mmol) and compound 3 (0.06 mmol) was added to the solution, which was reacted for another 36 h. The product, denoted as PCP-Mn-DTA, was collected by centrifugation  and vacuum drying.Mn-DTA (10 mg), 1-ethyl-3-(3-dimethyl amino propyl) carbodiimide hydrochloride  and N-hydroxysuccinimide  were dissolved in PBS . After stirring for 1.5 h, compound 3 was conducted similar to the synthesis of the PCP-Mn-DTA nanoparticles. Finally, the drug-loaded PCP-Mn-DTA@GOx@1-MT was harvested by centrifugation  and lyophilization.Mn-DTA (10 mg), GOx (1.33 mg) and 1-MT (2 mg) were dissolved in PBS (pH 7.4). After stirring at room temperature for 24 h, the mixture was centrifuged , followed by resuspension in PBS  containing EDC·HCl (15 mM) and NHS (15 mM). The conjugation of compound The loading of GOx and 1-MT in PCP-Mn-DTA@GOx@1-MT was quantified by UV-vis spectroscopy based on the standard curves of GOx at 276 nm and 1-MT at 288 nm, respectively. The drug loading content (DLC) and drug loading efficiency (DLE) were calculated by following equations:2O2 generation and pH change were monitored.In order to detect the activity of GOx loaded in the PCP-Mn-DTA@GOx@1-MT nanosystem, glucose (1 mg/mL) was mixed with free GOx (200 μg/mL) or an equivalent amount of GOx loaded PCP-Mn-DTA@GOx@1-MT (2.27 mg/mL). The supernatant was taken out at different time intervals , and the H2O2 or glucose, followed by transferring to the dialysis bags. The dialysis bags were subsequently immersed in PBS (29 mL) with different conditions under stirring (100 rpm) at 37 °C. The medium (0.7 mL) was taken out at desired time intervals , and the bags were refreshed with the same volume of PBS. The released GOx was measured by UV-vis spectroscopy at a wavelength of 276 nm.PCP-Mn-DTA@GOx@1-MT (3 mg) was first dissolved in PBS  without or with different concentrations of HBriefly, PCP-Mn-DTA@GOx@1-MT (1 mg/mL) was incubated with 10% fetal bovine serum (FBS) in PBS (pH 7.4 and 6.8) at 37 °C for 6 days. The sample solution was taken out at specific time intervals  and was detected by DLS to analyze the changes in the size of PCP-Mn-DTA@GOx@1-MT.2 at 37 °C.B16F10 melanoma cells were purchased from Cell Bank of Chinese Academy of Sciences, China. The cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% (w/v) penicillin (100 U/mL)/streptomycin (100 μg/mL) and 10% (v/v) FBS having 5% CO4 cells per square centimeter) seeded on the 24-well plates were treated with PBS, 1-MT (7 μM), PCP-Mn-DTA (11.36 μg/mL), PCP-Mn-DTA@1-MT, PCP-Mn-DTA@GOx@1-MT , and PCP-Mn-DTA@GOx@1-MT + glucose (5.5 mM glucose) for 24 h and 48 h, followed by incubating with the mixture solution composed of 200 μL fresh medium and 20 μL CCK-8 at 37 °C for another 1.5 h. The absorbance at 450 nm was recorded by a spectrophotometric microplate reader .To evaluate the cytotoxicity, the B16F10 cells  and PCP-Mn-DTA@GOx-FITC@1-MT at pH 7.4 and 6.8 for 4 and 12 h. The cells were subsequently fixed with 4% paraformaldehyde, permeabilized with 0.5% TritonX-100, followed by staining with ActinRed 555 and 4’,6-diamidino-2-phenylindole (DAPI). CLSM (LSM 510 META Olympus) and CellSens Dimension software were employed to study the efficiency of uptake by the B16F10 cells. On the other hand, the B16F10 cells treated with the various systems were washed with PBS. The cells were subsequently centrifuged (800 g × 10 min) at 4 °C and collected. Finally, they were resuspended in the cell binding solution (300 μL) and analyzed by FCM  and FlowJo_V10 software.Typically, DCF-DA was used as the ROS probe to monitor the ROS generation in the B16F10 cells. Briefly, the B16F10 cells seeded on the 6-well plates or confocal microscope dishes were treated with GOx, PCP-Mn-DTA, PCP-Mn-DTA@GOx, PCP-Mn-DTA@GOx@1-MT and PCP-Mn-DTA@GOx@1-MT + glucose with the same concentration of GOx. The cellular ROS generation was imaged by CLSM and quantitatively analyzed by FCM.19. Next, MCSs were transferred to two 6-well plates containing DMEM with different pH values, followed by treatment with the PCP-Mn-DTA@GOx@1-MT nanoparticles (11.36 μg/mL) labelled with FITC for 4 and 12 h. The resultant MSCs were washed by PBS, re-suspended in fresh medium and subsequently detected by CLSM.Firstly, hot agarose (1.5%) solution was poured into a 96-well plate (80 μL per well), which was cooled to form a layer of agaropectin. Next, MCSs were formed after incubating B16F10 cells on this plate about a weekBriefly, IDO1 -transfected B16F10 positive cells and B16F10 cells were incubated with PBS, PCP-Mn-DTA, 1-MT, PCP-Mn-DTA@1-MT, and PCP-Mn-DTA@GOx@1-MT for 24 h, respectively. Next, the cells were lysed and obtained by centrifugation . The western blotting analysis was used to measure the expression of IDO1.5 cells/well) were mixed with the T cells (5 × 105 cells/well) in a 24-well plate, followed by treating with 1-MT, PCP-Mn-DTA, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT, respectively. After 12 h co-culturing, the lymphocytes were obtained and incubated with EdU (10 μM) for the FCM analysis.IFN-γ-stimulated B16F10 cells  and Lysotracker red (1 μM) at 37 °C for 1, 3, and 8 h. Afterwards, the cells were then washed with PBS and observed by CLSM.Once the confluence of the B16F10 cells seeded in a 6-well plate reached 60–70%, the cells were treated with PBS (Control), 1-MT (7 μM), PCP-Mn-DTA (11.36 μg/mL), PCP-Mn-DTA@1-MT, PCP-Mn-DTA@GOx@1-MT  and PCP-Mn-DTA@GOx@1-MT + glucose (5.5 mM glucose) for 24 h. The cells were subsequently obtained by centrifugation (800 g), and stained by the Annexin V-FITC/PI kit (NeoBioscience) according to the instructions, followed by the FCM analysis.B16F10 cells seeded on the 6-well plates were treated with PBS, 1-MT (7 μM), PCP-Mn-DTA, PCP-Mn-DTA@1-MT, PCP-Mn-DTA@GOx@1-MT  and PCP-Mn-DTA@GOx@1-MT + glucose for 24 h, respectively. Afterwards, the cells were lysed, and the protein samples were collected through centrifugation . Western blotting  and ImageJ software 1.45 f were used to monitor and analyze the expression of Bax, Bcl-2 and Cyt-C .5  cells. For the invasion experiment, 2 × 105 B16F10 cells were cultured on the Transwell pre-covered matrix gel. Meanwhile, a medium containing 10% FBS was added to the lower chamber as a chemical attractant. After incubation for 24 h, the cells on the lower surface of Transwell were stained by crystal violet. They were subsequently counted and imaged by using a microscope.The B16F10 cells were treated with the different systems used herein for 24 h. Later, the cells were digested and collected by centrifugation (800 g). For the cell migration experiment, the cells were resuspended in a serum-free medium and subsequently transferred to the upper chamber of Transwell at a density of 1 × 106) in PBS into the right flank of the C57BL/6 or Balb/c mice19. Once the tumor volume reached about 50 mm3, seven groups of the tumor-bearing mice were intravenously injected with saline, Mn-DTA, PCP-Mn-DTA, GOx, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT at a dose of 3 mg/kg 1-MT (n = 6). These treatments were operated twice per week and lasted for 18 days. The tumor volumes and body weights of mice were measured every 2 days. The tumor volume (V) was calculated by using the equation:C57BL/6 mice and Balb/c mice (about 6 weeks old) were purchased from Beijing Institution for Drug Control (China). All animals were bred in the pathogen-free facility with a 12 h light/dark cycle and relative humidity (40–70%) at 21 ± 2 °C. All the mice had access to food and water ad libitum. The animal studies were conducted according to the guidelines of the Institutional Animal Care and Use Committee at Northwestern Polytechnical University. The tumor-bearing mouse models were established by inoculating B16F10 or 4T1 cells  and CD8+ T cells (CD3+CD4−CD8+), co-marked with anti-CD3-APC/Cy7 and anti-CD49b-PE/Cy7 for analyzing the NK cells (CD3−CD49b+), co-marked with anti-CD3-APC/Cy7 and anti-CD45R/B220-PE for analyzing the B cells (CD3−CD45R/B220+), co-marked with anti-CD80-APC, anti-CD86-PE and anti-CD11b-FITC for analyzing the mature dendritic cells (CD11b+CD80+CD86+), and co-marked with anti-CD4-FITC, anti-Foxp3-Alexa 647, and anti-CD3-APC/Cy7 for the analysis of the Treg cells by using FCM, according to the procedures of the manufacturer.The B16F10 and 4T1 cell-bearing mice were intravenously injected with the same seven formulations. The injections were operated twice every 2 days. Subsequently, the tumor tissues were excised, digested and harvested into mono-dispersive lymphocytes. The obtained lymphocytes were co-stained with anti-CD4-FITC, anti-CD3-APC/Cy7, and anti-CD8-Percp/Cy5.5 antibodies for analyzing the CD4The splenocytes collected from the treated mice were marked with anti-CD44-FITC, anti-CD3-PerCP-Cy5.5, anti-CD62L-APC, and anti-CD8-PE antibodies according to the procedures of the manufacturer. Afterwards, the detection with FCM was carried out to measure the central and effector memory T cells.The B16F10 cell-bearing mice were intravenously injected with the formulations every 3 days for 5 times. The tumor samples were harvested, homogenized, centrifuged and measured by using high performance liquid chromatography-mass spectrometry (HPLC-MS).Tumor-bearing mice were intravenously injected GOx or PCP-Mn-DTA@GOx@1-MT for various time intervals. The photoacoustic signals of oxygenated hemoglobin, hemoglobin and melanin in tumor sites were detected with iThera Medical MSOT inVision 128  and analyzed with ViewMOST (Release 3.8.1.09) software.The mice were sacrificed after the treatment for 18 days. The spleen, kidney, lung, heart, liver, and tumor were resected and made into sections. For the H&E assay, the sections of the tissues were stained with H&E and imaged by using a microscope  with CellSens Entry software. For the TUNEL assay, the tumor sections were stained with the TUNEL agents (Beyotime) and imaged by a CLSM. For the IFC study, the tumor sections were blocked by 5% bovine serum albumin and incubated with Ki67, IDO, mTOR, or STAT3 antibody. Afterwards, they were stained with Alexa Fluor 488-labeled IgG or Cy3-labeled second antibody according to the manufacturer’s instructions . Finally, the sections were stained with DAPI and characterized using a CLSM.PCP-Mn-DTA@GOx@1-MT along with control  were intravenously injected into the mice. After 1 h of administration, the blood was collected for the blood glucose determination. The blood glucose level of the mice was measured by using a blood glucose meter before and after the administration. Additionally, the blood of the mice was collected directly from the eyes after the last treatment. The blood samples were stored at 4 °C overnight and centrifuged at 900 g for 20 min to separate the plasma. Subsequently, the blood biochemical levels and hematological parameters as well as the liver and kidney function indices were determined according to the protocols recommended by the manufacturers.The B16F10 tumor-bearing C57BL/6 mice were intravenously injected with free 1-MT and PCP-Mn-DTA@GOx@1-MT at an equivalent 1-MT dose of 3 mg/kg. For pharmacokinetic study, at the desired time intervals, the blood was collected from the orbital plexus and heparinized. The plasma was harvested by centrifugation (2000 g). Acetonitrile was then added to precipitate proteins, followed by centrifugation at 18,500 g for 8 min to collect the supernatant. Finally, the collected sample was dried, re-dissolved and detected by HPLC. For biodistribution study, the mice were sacrificed after 24 h, and the tumor and major organs were collected and homogenized in DMSO (0.5 mL), followed by centrifugation at 16,000 g for 15 min. The amount of 1-MT in these organs was determined by HPLC.n = 6) were intravenously injected with saline (control), Mn-DTA, PCP-Mn-DTA, GOx, 1-MT, PCP-Mn-DTA@1-MT and PCP-Mn-DTA@GOx@1-MT on day -6, -4 and -2. The primary tumors were surgically resected on day 0, and the mice were subcutaneously re-challenged with the 4T1 or B16F10 cells and monitored for the recurrence of the secondary tumors.After primary tumor inoculation, the B16F10-bearing C57BL/6 mice and 4T1-bearing Balb/c mice  by the Student’s t-test and one-way analysis of variance (ANOVA). The data were expressed as means ± standard deviation (SD). The confidence levels of 95% and 99% were regarded as the significant difference.Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Exendin-4 (Ex-4), better known in its synthetic form and used clinically as exenatide, currently applied in the treatment of diabetes, induces a beneficial impact on nerve cells, and shows promising effects in obstructive lung diseases. At an advanced age, the development of the neurodegenerative process of brain tissue is masked by numerous concomitant diseases. The initial latent phase of neurodegenerative disease results in occurrence of manifestations at an advanced stage. To protect the brain and to simultaneously ensure proper treatment of common coexisting conditions in late life, such as diabetes, chronic obstructive pulmonary disease, or asthma, a pleiotropic medication should be chosen. Molecular mechanisms of Ex-4 exert neuroprotective effects or lead to secondary neurogenesis. Additionally, Ex-4 plays an important role in anti-inflammatory actions which are necessary both in the case of asthma and Parkinson’s disease. Specific receptors in the lungs also reduce the secretion of surfactants, which decreases the risk of exacerbation in chronic obstructive lung disease. In a great number of patients suffering from diabetes, asthma, or chronic lung disease, there is a great potential for both treatment of the main condition and protection against brain neurodegeneration. Extended life expectancy has resulted in an increased interest in age-specific conditions, such as Parkinson’s and Alzheimer’s disease. The growing interest in these diseases affecting the geriatric population results in numerous studies that search for therapies of neurodegenerative disorders, using the mechanisms of Exendin-4 (Ex-4).The latest publications on the effects of Ex-4 therapy clearly indicate that this drug could be used in the treatment of neurodegenerative diseases . Some ofPatients with Alzheimer’s disease are another target group. Currently, 5–7 million new cases of Alzheimer’s disease are diagnosed annually . NeurodeNumerous studies, driven with various material , lead to the same conclusion: not only diabetes , which hHeloderma suspectum) [2-Asp3 with Gly2-Glu3 causes differences in the bioactivity of these proteins and a significant reduction in the ability of Ex-4 to interact with receptors for proteins from the family of vasoactive intestinal proteins (VIP). This change does not affect the ability to bind to glucagon-like protein-1 receptors (GLP-1R) [Exendin-4 (Ex-4) was isolated from the saliva of the venomous lizard, Gila monster (spectum) . It is c(GLP-1R) . The seq(GLP-1R) , and sec(GLP-1R)  , an enzyme responsible for degradation of GLP-1 and Ex-4 . As a reAn analysis of the most common application of Ex-4, the mechanisms of action used in T2DM therapy, receptor localization, factors favouring receptor binding, and distribution throughout the body suggest other potential applications of Ex-4 whose effects have been confirmed in studies on animal models  or in in vivo cell cultures.Currently, apart from its use in diabetes, many neuroscientists are working on the application of Ex-4 in the therapy of neurodegenerative diseases. This is evidenced by numerous studies on the use of Ex-4 in the treatment of Parkinson’s disease. In 2017, the Institute of Neurology and Neurosurgery in London published results of its randomized, double-blind trials conducted on patients with Parkinson’s disease [Theoretical assumptions described in the above-mentioned research, as well as in many other studies, are based on the hypothesis that Ex-4 prevents or inhibits the neurodegenerative process, which can be divided into several elements. The first of them is the neuroprotective effect. This is achieved by preventing the activation and expression of metalloproteinase-3 (MMP-3) in the substantia nigra of the mesencephalon and striatum. Any action or medication which stops or inhibits its expression promotes the reduction in dopaminergic neurodegeneration. This neuroprotective property more effectively prevents the activation of microglial cells in the mentioned structures of the encephalon . Kim S. Moreover, an increase in the number of neuronal microtubules associated with protein 2, β-III-tubulin, and neuro-specific enolase with concomitant increase in the number of stem cells promotes neurogenesis even in adult individuals, which leads to further normalization of dopamine imbalance . SignifiIn the course of Ex-4 therapy, an increase in the phosphorylated form of cofilin in nerve cells is observed. Phosphorylation of this protein, i.e., its deactivation, inhibits apoptosis induction, which results in the reduced mortality of these cells . Moreove2O2 [Ex-4 therapy appears to be beneficial for patients with neurodegenerative diseases, characterized by the degeneration of cholinergic neurons, such as amyotrophic lateral sclerosis (ALS). In this disease, the administration of Ex-4 reduces the loss of vitality and permeability of the cell membrane induced by H2O2 . Along w2O2 . Secondary neurogenesis, which occurs after the completed development of nerve tissue in adults, is another important mechanism of action of Ex-4 which is involved in the inhibition or prevention of neurodegeneration. Ex-4 promotes the above process by increasing the number of microtubules associated with protein 2, β-III-tubulin, and neuro-specific enolase , as ment2O2 toxicity, inducing oxidative stress. This reduction may be achieved by deactivation of enzymes, i.e., phosphorylation of cofilin which induces apoptosis [2O2 [The third element is the ability of Ex-4 to reduce oxidative stress toxicity in nerve tissue. An animal model study (18-day Sprague-Dawley rat embryos) reconfirmed 73% mortality of primary hippocampal cells after using glutamate. Additionally, after administration of Ex-4, 25% of cells were affected by apoptosis. It follows that the other 75% were protected and stayed alive . Furtherpoptosis  or prolosis [2O2 .The above-mentioned mechanisms justify the growing interest in Ex-4. Many studies deal with the implementation of the drug into therapy or prevention of neurodegenerative diseases. Various locations of the Ex-4 receptor encourage a search for further action profiles, apart from the one that can be used in neurology.Hypoxia resulting from impaired blood supply or lack of blood supply to the heart, brain, kidneys, or the pancreas affects tissue metabolism. Hypoxia can dysregulate gene expression, such as HIF-1α , or contAfter the administration of Ex-4, hypoxic cells demonstrate substantially decreased activation of HIF-1α. Additionally, decreased expression of Bcl/Bax genes inhibits apoptotic pathways. Reduction in apoptosis following a prior Ex-4-induced reduction in HIF-1α expression indicates potential proapoptotic properties of this factor . The effIschemia causing hypoxia and re-reperfusion of cerebral tissue also contributes to a change in cellular immunoreactivity. It starts to intensify after only six hours following the change in conditions and reaches its extreme value as soon as after one day. On the next day, it starts to decrease, and on the fourth day it becomes the least intensive. Its value stays the same for next few days. This leads to delayed neuronal death. It is only after ten days that the immunoreactivity of brain tissue cells gets back to normal. The exact mechanism of this phenomenon has not been explained. Application of Ex-4 before the episode of ischemia significantly reduces hyperactivity induced by the drug on the first day, which proves its protective effect on nerve tissue. Implementation of such a therapy also considerably reduces the activation of the microglia in the ischemic region . The heart is another human organ which may suffer severe ischemia-related complications. A study conducted on cardiomyocytes confirms reports on the protective action of Ex-4 against damage caused by hypoxia. It does not only weaken hypoxia-induced apoptosis but also enhances intracellular expression of p38 mitogen-activated protein kinase (p38MAPK). This change is also accompanied by translocation of the glucose-4 transporter (GLUT-4). Ex-4 administration is associated with improved glucose uptake and increased production of ATP. Changes observed after application of Ex-4 include diminished cell destruction, which, consequently, results in cell viability, decreased LDH and creatine kinase (CK-MB) as well as reduced mortality. In addition, optimization of cell metabolism was noted .Mechanisms inhibiting apoptotic processes play an important role in prevention of neurodegeneration and protection in hypoxic conditions. The mechanisms include, among others, glutathione peroxidase (GPxR) and thioredoxin reductase (TRxR). Their increased activity reduces generation of reactive oxygen species (ROS) even by 50%. Moreover, it prevents reduction in protein kinase b activity (AKT) and allows the maintenance of its level prior to the episode of ischemia .Renal parenchyma tissue serves as a place where enhanced mRNA expression for anti-inflammatory factors, such as interleukin-10 (IL-10) and nitric oxide synthase (eNOS), occurs. Apart from various mechanisms of the GLP-1 receptor stimulation, a variable distribution of the receptor is observed. The different distribution depends on specific conditions. GLP-1R has an ability to autoregulate the expression after an acute ischemic episode. Moreover, there is a reverse correlation between the severity of kidney damage and expression of GLP-1 receptors in its parenchyma. This phenomenon stops deterioration of the renal function in the most severe cases. This is achieved by inhibition of macrophage recruitment, DNA damage, inflammation, and further production of ROS and other mechanisms inducing apoptosis, which intensifies the progress of kidney damage .Having followed the publication of Waddell et al.  and AhmaConsidering the definition and the underlying pathomechanisms of asthma, it appears that Ex-4 may be effective in the treatment of the disease. The mechanism described in the previous paragraph shows that Ex-4 weakens the expressions of TNF-α and IL-1β . A 12-weAnother reason for the use of Ex-4 in obstructive lung diseases is its ability to prevent bronchial hyperresponsiveness. In the course of various forms of asthma, patients often complain about triggering factors causing dyspnoea, which significantly reduces their daily functioning and decreases their quality of life. As shown in studies conducted on human bronchi under ex vivo conditions, GLP-1R stimulation leads to bronchial relaxation through the activation of cAMP protein kinase A (PKA) ,50,51. TCombining the identified mechanisms of Ex-4 with those already used in the treatment of asthma would allow the intensification of the effects of anti-inflammatory drugs and reduce the severity of symptoms by decreasing bronchial hyperreactivity ,51.Exposure to cigarette smoke or air pollution achieves a significant level not only in the big cities at present. Both contribute main risk factors to the development of chronic obstructive pulmonary disease (COPD) which is characterized by high mortality .Research conducted on an innovative experimental animal model of lung diseases  undergoing Ex-4 therapy resulted in a significant reduction in the mortality of mice with COPD through the induction of bronchial relaxation, not only by relaxing smooth muscles through GLP-1 but also by myorelaxation of the muscle layer in the vascular bed . It was 2+, which probably excludes the previously described mechanism of surfactant formation involving Ca-CM-PK [In an in vitro experiment on type II pneumocytes taken post-mortem from donors prepared for transplantation, the mechanism by which surfactant secretion could be regulated  was inveCa-CM-PK . Having Ca-CM-PK . In anotCa-CM-PK  analysinCa-CM-PK . RepeatiReduction in the expression of surfactant proteins, leading to a decrease in surfactant production and, as a consequence, to a decrease in the retention of surfactant proteins, may improve conditions for oxygen exchange in alveoli. It creates an opportunity to improve the normally already difficult oxygen shipment and to prevent the bacterial exacerbations in COPD. An enlarged vascular bed will be better supplied with blood which is responsible for oxygen transportation. Such a situation improves conditions of mechanisms reducing inflammation and oxaemia in the patient’s whole body. Reduced retention of sputum is also an important factor changing the conditions inside the alveoli in bacteriological terms. It is vital for prevention and treatment of bacterial infections in these patients, and often difficult to treat due to drug resistance.The results of the publications presented above are the evidence of possible applications of Ex-4 in the treatment of age-related diseases  Figure . StudiesThe studies also focused on the influence of Ex-4 on inflammatory markers such as TNF-α, IL-1β, NF-κB, SOCS-3, JNK-1, TLR-2, and TLR-4. Experiments confirming the location of GLP-1 receptors in the lungs and clinical trials carried out on the above models of lung diseases open new therapeutic possibilities for another field of medicine which could benefit from the described mechanisms of Ex-4 action. Currently, Ex-4 preparations administered in patients due to diabetic indications are being observed for their beneficial effect on the lungs. The described results will turn out to be key information in further clinical trials, confirming the effectiveness of lung disease treatment with Ex-4.Mechanisms triggered by Ex-4 active in hypoxia are also important. Depending on the time of its administration, the drug protects or minimizes damage caused by this traumatizing tissue condition.In light of population aging and multimorbidity, in the near future this exogenous equivalent of GLP-1 may become a valuable and important drug due to its multidimensional mechanisms of action. Introduction of Ex-4 into therapy will allow the triggering of regenerative processes, especially in brain tissue, as well as neuroprotective processes which are important in old age due to the accompanying burden of neurodegenerative and cardiovascular diseases . The mechanisms responsible for gene transrepression of the above-mentioned inflammatory factors reduce the inflammation which is often chronic in people affected by many concomitant diseases. Additionally, the drug improves regeneration or reduces damage to organs affected by hypoxia. The variety of effects of Ex-4 may prove to be crucial in the treatment of patients with many coexisting diseases that are often challenging for clinicians.


Traditional fossil-based plastic usage and disposal has been one of the largest environmental concerns due to its non-biodegradable nature and high energy consumption during the manufacturing process. Poly(lactic acid) (PLA) as a renewable polymer derived from natural sources with properties comparable to classical plastics and low environmental cost has gained much attention as a safer alternative. Abundantly generated orange peel waste is rich in valuable components and there is still limited study on the potential uses of orange peel waste in reinforcing the PLA matrix. In this study, orange peel fine powder (OPP) synthesized from dried orange peel waste was added into PLA solution. PLA/OPP solutions at different OPP loadings, i.e., 0, 10, 20, 40, and 60 wt% were then casted out as thin films through solution casting method. Fourier-transform infrared spectroscopy (FTIR) analysis has shown that the OPP is incorporated into the PLA matrix, with OH groups and C=C stretching from OPP can be observed in the spectra. Tensile test results have reviewed that the addition of OPP has decreased the tensile strength and Young’s modulus of PLA, but significantly improve the elongation at break by 49 to 737%. Water contact angle analysis shows that hydrophilic OPP has modified the surface hydrophobicity of PLA with a contact angle ranging from 70.12° to 88.18°, but higher loadings lead to decrease of surface energy. It is proven that addition of OPP improves the biodegradability of PLA, where PLA/60 wt% OPP composite shows the best biodegradation performance after 28 days with 60.43% weight loss. Lastly, all PLA/OPP composites have better absorption in alkaline solution. Plastic is a durable, inexpensive, and lightweight polymer that has a broad range of applications ranging from food packaging to electronic goods. Over 381 million tonnes of plastic are produced annually, and it was reported that around 55% was for single-use purpose only, 25% was incinerated, and 20% was recycled . ShrinkiDue to the increasing concerns on plastic pollution, biopolymer has attracted extensive interest in recent studies ,10. The Furthermore, citrus waste disposal has been challenging as it is generated abundantly but underutilized. According to Raimondo et al., the volume of citrus fruits processed is nearly 31.2 million tons per year, while 50 to 60% of the original mass become the residue ,19. The In this study, orange peel powder is incorporated into pure PLA to improve its biodegradability in natural environment while maintaining its good physical properties. Orange peel waste without prior chemical modification is converted into fine powder through mechanical grinding, then mixed at different concentrations with PLA in chloroform solution. The homogeneous solution is then casted onto flat surface to obtain smooth composite films through solution casting method. The biodegradability of the biocomposite film is studied by conducting soil burial degradation experiment over 28 days.PLA (IngeoTM biopolymer 3251D) was purchased from NatureWorks LLC . Sweet orange (Citrus sinensis) was purchased from local supermarket in Seri Iskandar, Perak. Chloroform (CAS 67–66–3) was purchased from R&M Chemicals . Hydrochloric acid (HCl) (CAS 7647–01–0) was purchased from Thermo Fisher Scientific . Sodium hydroxide (NaOH) was purchased from Sigma Aldrich . Deionized (DI) water was utilized throughout the experiment. The chemicals are utilized without further purification or treatment.Sweet orange (Citrus sinensis) peel collected was washed with tap water to remove impurities on the surface. Orange peel was dried under the sun for 20 h, followed by drying in oven for 18 h at 60 °C according to previous method by Farahmandfar . Dried oPLA pellets were dissolved in chloroform under constant stirring for 1 h at room temperature to make 10 wt% PLA solution. Around 0.5 g orange peel powder (OPP) was added into the PLA solution and stirred for another 1 h to prepare PLA/10 wt% OPP solution. The solution was casted onto flat glass plate and four petri dishes, respectively and dried for 24 h at room temperature, which is similar to experiment done by Musa et al., in synthesis of PLA/banana fibres film . The pro2 was placed on the FTIR. The intensity of transmitted light was computed in a wave range of 4000–500 cm−1. For water contact angle analysis, same size PLA film was mounted on goniometer. A single drop of deionized water was released from the syringe on top onto the specimen surface. The angle between the specimen surface and the edge of the water drop was measured. The mechanical strength analysis by using universal testing machine . Blank PLA film was prepared into sample of dumbbell shape following the ASTM D638 standard [The morphology of film was captured by using stereoscopic microscope (Leica S8 APO). The sample functional groups were characterized using Fourier Transform Infrared Spectroscopy (FTIR) Perkin Elmer Spectrum One. Blank PLA sample of size 2 × 2 cmstandard . Geometr2 were prepared from each composite and weighed to record respective initial weight as Wdry. Samples were immersed into three petri dishes containing different pH solutions, i.e., 0.1 M HCl, distilled water, and 0.1 M NaOH, respectively [swollen. The swelling index was calculated Equation (1) as belowThree samples with size of 4 × 4 cmectively . After 11). Each sample was buried under 2 cm of moist garden soil in a container and kept for 28 days in an open environment. The soil burial method is adapted from previous studies by Park et al., and Lertphirun et al., with some modification [2) of samples were recorded. The biodegradability of the composites was measured using Equation (2) as follows:Blank PLA and different concentrations of PLA/OPP composite samples casted in petri dish of diameter 8 cm were weighed to measure respective initial weight  is calculated by comparing the weight before and after immersing each composite sample in acid, alkaline, and neutral distilled water at pH 1, 13, and 7, respectively. Based on Soil burial experiment was conducted for a total period of 28 days, where the samples were taken out, weighed, and observed at every 7-day interval. The blank PLA film has shown the least weight loss throughout the soil burial experiment, with low percentage ranging from 3.73 to 5.49%. From −1) indicates the presence of hydroxyl group, which is only observed at PLA/OPP composites at higher loadings [−1 due to the presence of unsaturated aromatic compounds in orange peel [−1 indicates the presence of C-O stretching in all spectra [As observed from loadings . This isloadings ,34,35, a spectra ,37. Addi spectra . High su spectra , causingIt is believed that the cellulose components in OPP have helped in modifying the toughness by improving the elongation. The poor interfacial adhesion between cellulose fibres and polymer can cause agglomerations due to hydrophilic nature of OPP and hydrophobic nature of PLA, hence resulting in low tensile and modulus ,39. WhilFurthermore, the optimum loading is obtained at 20 wt% OPP that yields the highest elongation at break which gives the polymer a good ductility to not break easily. Nonetheless, at even higher OPP loadings (40 wt% and 60 wt%), all three mechanical properties, i.e., tensile strength, Young’s modulus, and elongation at break are found decreased again. This is probably due to the high OPP loadings that cause poor distribution and agglomeration of the filler particles, resulting in embrittlement of the composites. The mechanical strength results show similar agreement to study by Singh et al., whereas high loading of cellulose in PLA reduced tensile strength and modulus, while elongation at break was increased . The oraThe addition of OPP with hydrophilic cellulose fibre content has helped to bond the water molecules together through the affinity of OH groups, resulting in higher absorption or swelling rate ,45. CompAn increase in OPP loadings has led to better biodegradability of the composites due to the presence of higher amount of cellulose content. The high cellulose content (9.19 to 22%) in orange peel has favoured the biodegradation process through hydrolysis and microbial activities ,47. Hydr−1. Moreover, it was found that addition of OPP into PLA has generally decreased the tensile and modulus, but significant increase of elongation was observed at low loadings of OPP. It was suggested that the OPP which contains high cellulose fibre components modified the toughness and gave a better ductility to the composites. Water contact angle analysis has shown that the hydrophilic OPP significantly modified the surface hydrophobicity of PLA, with higher OPP loadings resulted in rough surface of composites and low surface energy. From soil burial test over a consecutive study of 28 days, it was concluded that the presence of OPP has improved the biodegradability of PLA. Better biodegradation was observed with increasing concentration of OPP.In this study, orange peel waste transformed into fine powder was incorporated into PLA to develop a biobased, biodegradable polymer. It was proven that OPP has been incorporated into the PLA matrix by the presence of hydroxyl group in the high energy region, and C=C functional group at wavenumber 1608 cm


Bisphenol A epoxy resin cured with a mixture of dimerized and trimerized fatty acids is the first epoxy vitrimer and has been extensively studied. However, the cure behavior and thermal and mechanical properties of this epoxy vitrimer depend on the epoxy/acid stoichiometry. To address these issues, epoxy vitrimers with three epoxy/acid stoichiometries  were prepared and recycled four times. Differential scanning calorimetry (DSC) was used to study the cure behavior of the original epoxy vitrimers. The dynamic mechanical properties and mechanical performance of the original and recycled epoxy vitrimers were investigated by using dynamic mechanical analysis (DMA) and a universal testing machine. Furthermore, the reaction mechanism of epoxy vitrimer with different epoxy/acid stoichiometry was interpreted. With an increase in the epoxy/acid ratio, the reaction rate, swelling ratio, glass transition temperature and mechanical properties of the original epoxy vitrimers decreased, whereas the gel content increased. The recycling decreased the swelling ratio and elongation at break of the original epoxy vitrimers. Moreover, the elongation at break of the recycled epoxy vitrimers decreased with the epoxy/acid ratio at the same recycling time. However, the gel content, tensile strength and toughness of the original epoxy vitrimers increased after the recycling. The mechanical properties of epoxy vitrimers can be tuned with the variation in the epoxy/acid stoichiometry. Epoxy resin, one of the most important and popular classes of thermosetting polymers, is firmly rooted in many areas, both in our daily lives and in industry ,2,3,4,5.v), resulting in a topology rearrangement under external force. Under this circumstance, vitrimers can flow like vitreous fluid [In 2011, Leibler et al. developed a new class of polymers, called vitrimer, based on associative covalent adaptable networks of epoxy resins cured with a mixture of dimerized and trimerized fatty acids through transesterification reactions . Vitrimeus fluid ,15. The us fluid ,17,18.Epoxy resins cured with carboxylic acids and anhydrides are the most studied epoxy vitrimers ,19,20. Dg) and bond exchange reactions of epoxy vitrimers [g and energy barrier of the vitrimer increase with the anhydride content in fatty acid and anhydride mixtures. Snijkers and coworkers compared the curing of epoxy vitrimers cured with three fatty acid mixtures [The long-chain dicarboxylic fatty acid can be obtained through the dimerization of fatty acids. Because of side reactions, the dimerized fatty acid is a mixture of monomers, dimers and trimers . Since 2itrimers . Both thmixtures . The actmixtures , the imp2) as the catalyst. The cure behavior of epoxy vitrimers was determined by differential scanning calorimetry (DSC). To investigate the effect of recycling on the solvent stability, thermal and mechanical properties of the original epoxy vitrimers, solvent immersion, dynamic mechanical analysis (DMA) and uniaxial tests were performed.In this work, the effect of the stoichiometry of epoxy and acid on vitrimer properties was investigated. Three epoxy vitrimers were prepared, using a commercial epoxy oligomer, a mixture of fatty acids with three stoichiometries (from 9:11 to 11:9 epoxy/acid ratio) as the curing agent and zinc acetate (Zn(Ac)2) was purchased from Shanghai Macklin Biochemical Co., Ltd. . Bisphenol A epoxy oligomer (trade name of 0164) was obtained from Nantong Xingchen Synthetic Material Co., Ltd. . A mixed fatty acid with about 23 wt% dimerized acid and 77 wt% trimerized acid was supplied by Shanghai Zhipu Chemical Co., Ltd. . Zinc acetate (Zn(Ac)2 was 10 mol% of the COOH groups. The epoxy oligomer was introduced into the masterbatch and stirred at 1500 rpm for 3 min at 120 °C. Finally, the mixture was quickly poured into a PTFE (polytetrafluoroethylene) mold with a diameter of 100 mm and a height of 5 mm and placed in an oven at 120 °C for 4 h. Zinc acetate was mixed with the mixture of fatty acids to prepare a masterbatch of curing agents in a 500 mL beaker at 900 rpm at 180 °C for 2 h. The content of Zn(Ac)Cured epoxy vitrimer film was cut into small pieces and then reprocessed to bulk material by the hot press at 180 °C and 10 MPa for 20 min. Then, the recycled sample was cut into small pieces and reprocessed again. The recycling process was repeated three times at a 10 °C increase in temperature each time. The reprocessed samples for the first, second, third and fourth times were abbreviated as 1R, 2R, 3R and 4R, respectively. 3 were mounted on a tension clamp and tested at a ramping rate of 3 °C/min and a frequency of 1 Hz from −25 to 125 °C. The Tg was determined as the temperature of the maximum damping factor (tan δ) value in a tan δ versus temperature curve. Isothermal and dynamic DSC analyses were performed on a DSC 1 instrument  under a nitrogen flow of 20 mL/min. For isothermal curing, the uncured sample (~20 mg) was heated rapidly to 120 °C and kept at that temperature until the heat flow leveled off to the baseline. The isothermal heat of reaction (ΔHi) was calculated by the integration of the exothermal curve in time. After the isothermal scan, the sample was cooled rapidly to 20 °C and then reheated to 300 °C at a heating rate of 10 °C/min to determine the heat of reaction during dynamic curing (ΔHt). The swelling ratio (SR) and gel content (GC) of cured networks were tested in toluene at room temperature for 48 h. After wiping the solvent, the swollen sample was weighed and dried at 80 °C for 24 h in a ventilated oven to remove the toluene. The SR and GC were calculated as follows:Tensile tests were conducted on an Instron 3366 testing machine according to ASTM D638 (type V). The dumbbell-shaped samples were tested at room temperature. The crosshead speed during measurements was 200 mm/min. At least five samples were measured for each composition. DMA was carried out on a DMA + 450 dynamic mechanical analyzer . The samples with dimensions of 30 × 20 × 2.5 mmDSC is a powerful tool for monitoring the cure reaction of epoxy resins ,37. To ddα/dt) is proportional to the heat flow (dH/dt) during a cure reactionα is the conversion (extent of reaction) and It is believed that the instantaneous reaction rate (As shown in The cure reactions of epoxides with acids in epoxy vitrimers are presented in To determine the solvent stability of the original and recycled epoxy vitrimers, samples were immersed in toluene at room temperature for 48 h. As shown in gs of the original and recycled epoxy vitrimers. The Tg of the original epoxy vitrimers decreases with the epoxy/acid ratio. A similar trend was reported in the tetrafunctional epoxy/dimerized acid vitrimer [g with the epoxy/acid ratio can be attributed to the crosslink density of epoxy vitrimers [g of the original epoxy vitrimers decreases with the epoxy/acid ratio.vitrimer . The varitrimers . As discg of the original vitrimer. In addition, except for the fourth recycling, the Tg of recycled vitrimers slightly decreases with the recycling time. However, EV55 shows an opposite trend since the occurrence of the catalytic ring-opening polymerization between epoxides occurs at a high recycling temperature, resulting in an increase in the crosslink density of the original epoxy vitrimer. For the recycled EV55, the Tg increases in the recycling time since the gradual increase in the recycling temperature brings about more catalytic ring-opening polymerization and, thus, a further increase in the crosslink density.For EV45, the recycling decreases the Tg and tensile strength. Therefore, the decrease in the tensile strength is attributed to the decrease in the crosslink density of the original epoxy vitrimers with the epoxy/acid ratio. The recycling significantly increases the tensile strength of the original vitrimers except for the first recycling. For the tetrafunctional epoxy/dimerized acid vitrimers, an opposite trend was shown [as shown . For theTensile toughness, the area under the stress–strain curve, indicates the energy absorption up to the material’s failure ,54,55. Tg of the original epoxy vitrimers decreases with the epoxy/acid ratio. The recycling decreases the Tg of the original epoxy vitrimer with the deficit epoxy oligomers. However, the Tg of the original epoxy vitrimer with the excess of epoxy oligomers increases after the recycling. In addition, the Tg of the recycled epoxy vitrimer with an excess of epoxy oligomers increases with the recycling time. Both the tensile strength and toughness of the original vitrimers increase with the increase in the epoxy/acid ratio and increase after the recycling. However, the elongation at break shows a contrary trend. Both the tensile strength and toughness of the recycled epoxy vitrimers with the stoichiometry and the excess of epoxy oligomers increase with the recycling time.This work investigated the effect of epoxy/acid stoichiometry on the cure behavior and mechanical properties of epoxy vitrimers. Epoxy vitrimers with three epoxy/acid stoichiometries from the deficit to the excess of epoxy oligomers were prepared and recycled. Because of the catalytic ring-opening polymerization at elevated temperature as well as the steric effect of long-chain fatty acids, the epoxy vitrimer with the excess of epoxy oligomers shows the lowest reaction rate and conversion during the isothermal curing. The swelling ratio of the original epoxy oligomers decreases with the epoxy/acid ratio, whereas the gel content shows a contrary trend. The recycling decreases the swelling ratio but increases the gel content in the original epoxy vitrimers. The swelling ratio of the recycled epoxy vitrimers decreases with the recycling time, while the gel content shows an opposite trend. For the epoxy vitrimer with an excess of epoxy oligomers, the recycling increases the storage modulus. The T


Morus alba L. has cytotoxic activity against several types of cancer cells. However, little is known about its chemopreventive mechanisms and bioactive metabolites. In this study, we showed that M. alba L. root bark extracts (MRBE) suppressed β-catenin response transcription (CRT), which is aberrantly activated in various cancers, by promoting the degradation of β-catenin. In addition, MRBE repressed the expression of the β-catenin/T-cell factor (TCF)-dependent genes, cmyc and cyclin D1, thus inhibiting the proliferation of RPMI-8226 multiple myeloma (MM) cells. MRBE induced apoptosis in MM cells, as evidenced by the increase in the population of annexin VFITC- positive cells and caspase-3/7 activity. We identified ursolic acid in MRBE through LC/mass spectrum (MS) and observed that it also decreased intracellular β-catenin, c-myc, and cyclin D1 levels. Furthermore, it suppressed the proliferation of RPMI-8226 cells by stimulating cell cycle arrest and apoptosis. These findings suggest that MRBE and its active ingredient, ursolic acid, exert antiproliferative activity by promoting the degradation of β-catenin and may have significant chemopreventive potential against MM.The root bark of  Multiple myeloma (MM), a malignant plasma cell neoplasm, is the second most frequent hematological malignancy, comprising 10–15% of all blood cancers . BecauseThe excessive accumulation of β-catenin and the subsequent upregulation of β-catenin response transcription (CRT) contribute to the development and progression of several cancers . In MM, 5Morus alba L. has been used in herbal medicine because of its anti-inflammatory, liver-protective, kidney-protective, hypotensive, diuretic, anti-cough, and analgesic effects [M. alba L. root bark extracts (MRBE) were shown to have anticancer activity [The root bark of  effects . Recentlactivity , 11 and activity . HoweverM. alba L. (1 kg) were extracted with 80% (v/v) ethanol (3 L) under reflux 3 times for 2 h. After the liquid was filtered, the fluid was concentrated under vacuum to obtain the extracts (9.7 g).The dried root barks of RPMI8226, HEK293, and Wnt3a-L cells were purchased from the American Type Culture Collection and cultured in RPMI1640 (RPMI8226 and MM.1S) and Dulbecco’s modified Eagle’s media (HEK293 and Wnt3a-L) supplemented with 10% fetal bovine serum, 120 μg/ml penicillin, and 200 μg/ml streptomycin. HEK293-FL (TOPFlash) reporter cells were established as previously described . Wnt3a-CCytosolic fractions were obtained as previously described . Whole-cTotal RNA was isolated using Trizol reagent (Invitrogen) following the manufacturer’s protocol. cDNA synthesis and PCR were performed as previously described . The ampCells were inoculated into 96-well plates and treated with either DMSO or MRBE (20 and 40 μg/mL) and ursolic acid (20 and 40 μM). After 48 h, the cell viability of each treated sample was measured in triplicate with the Celltiter-Glo Assay Kit (Promega) according to the manufacturer’s instructions. To calculate the inhibition of cell growth, the value at time 0 was subtracted.After treatment with either DMSO or MRBE (20 and 40 μg/ml) and ursolic acid (20 and 40 μM) for 48 h, cells were washed with cold phosphate-buffered saline and stained with annexin V-FITC and PI. The levels of apoptosis were then measured using the Apoptosis Detection Kit (BD Transduction Laboratories), according to the manufacturer’s protocols. Cells were then analyzed using a Cellometer cytometer .M. alba L. root bark metabolites were extracted as described previously [18 1.7 μm 2.1 × 150mm column  equipped with UPLC BEH C18 1.7 μm 2.1 × 5.0 mm VanGuard Pre-Column (Waters). The mobile phase consisted of 0.1% formic acid in water (buffer A) and 0.1% formic acid in acetonitrile (buffer B). The chromatographic separation was programmed as follows (buffer B): 0–2.0 min, 5%; 2.0–25.0 min, 5–95%; 25.0–27.5 min, 95%; and 27.6–30.0 min, 5%. The flow rate was set to 300 μL/min. The mass spectra data were acquired using Full MS-ddMS2 (top3) scan mode in positive ionization mode. Data acquisition and processing were performed using the Xcalibur software (Thermo Fisher Scientific).eviously , 18. An The activity of caspase-3/7 was determined in cell lysates using the Apo-ONE Homogeneous Caspase-3/7 Assay Kit (Promega).t-tests were employed to compare the means of the control and experimental groups. All experiments were conducted three times. Statistical significance was set to p < 0.05 (*) or p < 0.01 (**). The results were expressed as mean ± standard deviation (SD).Student's To elucidate the mechanisms underlying the MRBE anticancer activity, we used HEK293-firefly luciferase (FL) reporter cells stably harboring a synthetic β-catenin/TCF-dependent FL reporter (TOPFlash) and an hFz-1 expression plasmid. The incubation of HEK293-FL reporter cells with Wnt3a-conditioned medium (Wnt3a-CM) increased CRT activity. However, MRBE addition led to a concentration-dependent decrease in CRT . We thenGlycogen synthase kinase-3β (GSK-3β)-mediated β-catenin phosphorylation at Ser33/37/Thr41 is central in the proteasome-dependent β-catenin degradation pathway . TherefoA previous study demonstrated that β-catenin is constitutively upregulated in RPMI-8226 and MM.1S MM cells , we asseTo elucidate the antiproliferative mechanisms triggered by MRBE, we exposed RPMI-8226 cells to MRBE and determined the number of annexin V-FITC/propidium iodide-stained apoptotic cells using a cytometer. As presented in M. alba L., including terpenoids, flavonoids, and stilenoids [M. alba L.[+ ions . As shown in M. alba L., a traditional Chinese medicine, produces various natural compounds, including terpenoids, flavonoids, and stilenoids [Medicinal plants are potential resources for developing therapeutics targeting human diseases. The root bark of ilenoids . Interesilenoids , 12. HowThe Wnt/β-catenin pathway plays a significant role in regulating cell proliferation, differentiation, and development -31. CentConstitutive upregulation of the Wnt/β-catenin pathway frequently occurs in MM without the presence of mutations in this pathway . In addiBased on one-point estimation with the standard compound, the molar concentration of ursolic acid in 40 μg/mL MRBE was 0.13 μM, which is lower than the minimal effective concentration on MM cell growth inhibition . SeveralIn conclusion, this is the first study to identify the antiproliferative potency of MRBE and ursolic acid against MM cells and reveal the molecular mechanism. MRBE and ursolic acid accelerate oncogenic β-catenin turnover and repress β-catenin-dependent gene expression, thereby suppressing MM cell growth through apoptosis. Therefore, MRBE and ursolic acid have potential to be developed into preventive or therapeutic agents for MM treatment.http://jmb.or.kr.Supplementary data for this paper are available on-line only at