The Pt‐decorated MOF@GNSs (PtMGs) are further functionalized with human serum albumin‐chelated gadolinium  and loaded with indocyanine green (ICG) (ICG‐PtMGs@HGd) to achieve a synergistic PDT/PTT effect and fluorescence (FL)/multispectral optoacoustic tomography (MSOT)/X‐ray computed tomography (CT)/magnetic resonance (MR) imaging. The Pt‐decorated nanoplatform endows remarkable catalase‐like behavior and facilitates the continuous decomposition of the endogenous H2O2 into O2 to enhance the PDT effect under hypoxic TME. HSA modification enhances the biocompatibility and tumor‐targeting ability of the nanocomposites. This TME‐responsive and O2 self‐supplement nanoparticle holds great potential as a multifunctional theranostic nanoplatform for the multimodal imaging‐guided synergistic phototherapy of solid tumors.Multifunctional nanoplatforms for imaging‐guided synergistic antitumor treatment are highly desirable in biomedical applications. However, anticancer treatment is largely affected by the pre‐existing hypoxic tumor microenvironment (TME), which not only causes the resistance of the tumors to photodynamic therapy (PDT), but also promotes tumorigenesis and tumor progression. Here, a continuous O 2 self‐enriched drug delivery platform based on octahedral gold nanoshells (GNSs) using Pt‐decorated metal–organic frameworks (MOF) as inner template is constructed. The obtained nanostructures are further functionalized with human serum albumin‐gadolinium hybrid  and loaded with indocyanine green (ICG) (ICG‐PtMGs@HGd) to achieve a multimodal imaging guided enhanced photodynamic therapy/photothermal therapy PDT/PTT effect.In this research, a tumor environment (TEM)‐responsive and continuous O Photodynamic therapy (PDT) and photothermal therapy (PTT) are the two main phototherapies. PDT utilizes the photosensitizers (PS) to generate reactive oxygen species (ROS) under light irradiation to kill the tumor cells while PTT enables the tumor death via the local hyperthermia induced by the photothermal agents under light irradiation. Multifunctional nanoparticles enabling the combinatorial effects of PDT and PTT, and the simultaneous in situ imaging such as the optical imaging, photoacoustic tomography (PA), X‐ray computed tomography (CT), and magnetic resonance imaging (MRI) have been constructed. However, the anticancer therapy of such high‐performance nanoplatforms remains intractable challenging due to the complex tumor microenvironment (TME) and the induced therapeutic resistances.Nanotheranostic platforms that combine the multiple antitumor therapies under the guidance of multimodal imaging have become the trend in cancer therapy due to their enhanced therapeutic effect, improved tumor specificity, reduced drug resistance, and the convenience for the precise tumor lesion detection and treatment monitoring.1 With Hypoxia not only caused the resistance of the tumors to PDT that requires high concentration of oxygen to generate the cytotoxic ROS, but also promoted the tumorigenesis and tumor progression as a result of DNA methylation and the high expression of hypoxia‐inducible factors (HIFs). Meanwhile, the unexpected tumor metabolites such as hydrogen peroxide (H2O2) are also generated under the hypoxia condition, which promotes the invasiveness and metastasis of cancer. Many efforts have been devoted to alleviate the hypoxia and to improve the PDT effect, including the hyperbaric oxygen therapy, the oxygen carriers  and the catalyze‐like oxygen generators  and copper oxide (CuO)). Among them, utilizing the nanozyme as catalysts to generate oxygen in situ, has attracted increasing interests due to its resistance to the stringent conditions, the tunable enzymatic activity, and the low production cost. Many noble metal nanomaterials have been used as catalytic nanozymes for oxygen generation to enhance ROS production in TME, among which platinum (Pt) and palladium (Pd) nanoparticles exhibit durable catalytic and antioxidant properties and serves as a desire catalyst to continuously decompose H2O2 into O2 without rapid self‐consumption as was common for the other catalysts such as MnO2.Hypoxic microenvironment is one of the typical characteristics of the solid tumors owing to the imbalance between the high oxygen consumption caused by the fast proliferation of the tumor cells and the inefficient supply of oxygen caused by the malformation of the tumor vascular systems.5 Hypo2 self‐supplement nanoplatform to achieve an improved synergistic phototherapy with the guidance of FL/MSOT/CT/MR quadruple‐modal imaging based on Pt‐decorated gold nanoparticles using metal–organic frameworks (MOFs) as the inner template  have been widely applied in PTT owing to their surface plasmon resonance (SPR) ability and the porous structures for the synergistic therapy with other modalities. The GNSs also possess ideal X‐ray attenuation coefficient, holding great potential for CT imaging. The conventional method for preparing GNSs is to deposit the gold materials onto the surfaces of the suitable templates, such as the mesoporous silica and the metal nanoparticles , which requires the complicated synthetic procedure and the strict reaction conditions. In this regard, the tailorable synthesis and the tunable pore aperture of MOF make it a promising template to construct GNSs. In our design, the porous GNSs were constructed by a rapid and straightforward one‐step method in which the gold was grafted on the template of Pt‐loaded MOFs . The obtained PtMGs were further decorated with human serum albumin (HSA)‐gadolinium (Gd) hybrids  (PtMG@HGd) to enable MR imaging utilizing the high longitudinal proton relaxivity of Gd. Meanwhile, the coating of endogenous HSA that is approved by the U.S. Food and Drug Administration (FDA) for intravenous administration increases the biocompatibility, stability, and tumor targeting effect (passive or active) of the nanoplatforms. Finally, indocyanine green (ICG), a commercially NIR organic dye that has been approved by FDA is loaded into the nanoplatform, endowing the nanocarriers synergistic PDT/PTT effect as well as fluorescence (FL)/multispectral optoacoustic tomography (MSOT) imaging.Here, we develop a hypoxia‐resistant and persistent Oe Scheme 1. MOFs,The as prepared ICG‐PtMG@HGd nanocomposites enable persistent regulation of the hypoxia microenvironment in the tumor by the Pt‐induced self‐supply of oxygen, and exhibit improved effects in the oncolysis and metastasis inhibition of the tumor by the synergistic phototherapy. The simultaneously obtained FL/MSOT/CT/MR quadruple‐modal imaging provides more accurate information of the tumor from the optical, electronic, and magnetic perspectives. This versatile nanotheranostic platform shows its advantages in the treatment and in situ imaging of the tumor, provides insights into design of the multifunctional nanoparticles for TME‐responsive cancer therapy.22.1Figure 4, and the organic ligands  to form amino modified Pt‐MOFs (PtMs). Transmission electron microscope (TEM) images showed that the Zr‐based MOFs maintained regular 3D octahedral structure, and the uniform size and dispersion after Pt doping  studies  by anchoring the gold seeds from the overnight‐aged HAuCl4–K2CO3 solution to the amino groups on the surface of PtMs via electrostatic adsorption followed by local reduction in polyvinyl pyrrolidone (PVP) and a mild reductant . This one‐step synthesis method enabled rapid, facile, and straightforward coating of the porous gold nanoshells outside the nanoparticle cores. TEM images revealed a relative uniform layer of gold nanoshells coated on the surfaces of PtMs and the morphological structures remained almost unchanged  compared to the intrinsic MOFs (927.14 m2 g−1) was mainly due to the contribution of nonporous Pt nanoparticles and the existence of PVP in the composites. Taken together, these results demonstrated that the NH2‐modified Pt‐MOFs could serve as effective templates for the coating of the porous gold nanoshells by a facile and versatile method.The octahedron porous gold nanoshells were fabricated using Pt encapsulated MOF as the inner template Scheme . The Pt s (PtMs).26 Trag Figure ‐II,III, s Figure , which sd Figure ‐IV. Highd Figure . These rd Figure  indicated Figure . We perfd Figure ‐I was la The T1‐weighted MR imaging capacity and the corresponding signal intensity of ICG‐PtMGs@HGd exhibited a relatively high value among existing Gd‐based contrast agents , further confirming that the HGd hybrids was anchored to PtMGs@HGd. In addition, the molar ratio of Zr, Pt, Gd, and Au in PtMGs@HGd was detected to be 9.2, 3.2:1.8:100 by ICP‐MS, guaranteeing the H2O2 catalytic effect and MR imaging of the nanoplatform owing to the abundant loading content of Pt and Gd.The obtained PtMGs were further decorated with HGd and loaded with ICG to establish the versatile PtMGs@HGd nanoparticles for the multimodal imaging‐guided cancer therapy. PtMGs nanoparticles were first decorated with 11‐mercaptoundecanoic acid (MUA) by Au—S bond to obtain PtMGs–COOH. After that, HSA‐Gd hybrids were covalently connected onto PtMGs‐COOH nanoparticles via carbodiimide‐catalyzed amidation reaction Scheme  and the 30 The In the UV–vis spectra, the absorption of ICG‐PtMGs@HGd showed a characteristic peak at 800 nm with a slight red shift and a wider absorption compared with the free ICG ). Visual photographs of the samples after 72 h showed that the ICG‐PtMGs@HGd nanoparticles were well dispersed without aggregation in all the media . We also measured the particles sizes of the samples in different media at different time points  by DLS, and observed no obvious changes in all the groups over 72 h , indicating the high stability of the nanocarriers in different physiological solutions.2.22O2 consumption assay using titanium sulfate (Ti(SO4)2) as a colorimetric indicator. The UV–vis absorption spectra of H2O2–Ti(SO4)2 decreased with time after the treatment with ICG‐PtMGs@HGd and the calculated degradation ratio of H2O2 showed that about 70% of H2O2 was consumed within 240 min after the addition of ICG‐PtMGs@HGd  as an probe. As seen in Figure 1O2 in PBS and PtMGs@HGd solutions after light irradiation, while the production of 1O2 significantly increased when ICG was loaded (ICG‐PtMGs@HGd), demonstrating the PDT effect of ICG, which was in accordance with the previous reports showing that ICG could not only serve as an NIR dye for fluorescent and photoacoustic imaging, but could also generate ROS and hyperthermia for PDT and PTT. The generation of 1O2 from ICG‐PtMGs@HGd after light irradiation significantly deceased under hypoxic condition, but could be obviously enhanced when adding H2O2. However, this H2O2‐induced enhancement of 1O2 production was not observed in the ICG‐MGs@HGd nanoparticles without Pt nanozyme, further verifying the Pt nanozyme‐induced catalytic activity and the resulting improved PDT effect of ICG‐PtMGs@HGd under hypoxic atmosphere and mimetic H2O2 environment.After confirming the effective Han probe.34 As 2O2‐pretreated 4T1 cells were incubated with the ICG‐PtMGs@HGd dispersion at low (45 µg mL−1) and high (90 µg mL−1) concentrations, and MAK164, an H2O2 probe, was used to estimate the decomposition of intracellular H2O2. We found that the fluorescent intensity of the cells significantly decreased in a concentration‐dependent trend after incubation with ICG‐PtMGs@HGd 3]Cl2 whose fluorescence could be quenched after reacting with O2 molecule. 4T1 Cells treated with ICG‐PtMGs@HGd exhibited decayed red fluorescence in a concentration‐dependent manner compared with the ones without ICG‐PtMGs@HGd treatment under hypoxic condition  was used as the hypoxia indicator to evaluate the catalytic activity of ICG‐PtMGs@HGd since the hypoxic cells are characterized by intracellular accumulation of HIF‐1α. We used anti‐HIF‐1α antibody and rhodamine‐conjugated phalloidin to costain HIF‐1α and tubulin in 4T1 cells under normoxia and hypoxia conditions. As expected, the cells under normoxic condition showed no expression of HIF‐1α (green), while the cells under hypoxic condition exhibited significant accumulation of HIF‐1α  of the ICG‐PtMGs@HGd nanoparticles was determined to be 38.55% , which was higher than the conventional gold nanoshells. This may be attributed to the unique octahedral structure of the MOF‐based nanoparticles in which the multiple sharp points can effectively make the nanoparticles capture more laser energy.We then explored the photothermal performance of ICG‐PtMGs@HGd. Exposed to laser irradiation for 5 min, PtMGs@HGd exhibited a concentration‐ and irradiation time‐dependent temperature increased characteristics . We also performed the DLS experiment of the PtMGs@HGd nanocarriers before and after laser irradiation, and found their size distribution and zeta potential kept well after laser irradiation , suggesting the favorable stability of the nanoparticles when irradiated by laser. We next compared the PTT effect of different formulations, and found that ICG‐PtMGs@HGd possessed significantly higher thermal properties compared to PtMGs@HGd and free ICG Figure , which wnoshells.1, 42 generation during therapeutic treatment. As shown in Figure S11 (Supporting Information), the increased oxygen of PtMGs@HGd (from 4.64 to 11.55 mg mL−1) and PtMGs@HGd + Laser (from 4.80 to 11.41 mg mL−1) had no obvious difference, demonstrating the stability of Pt‐catalyzed effect is excellent and the oxygen generation could not be affected by the photothermal effect.We further investigated whether photothermal effect can enhance the Pt catalyzed O2.4 we utilized LysoTracker Green, a lysosome binding dye, to ascertain the localization of the lysosomes and the nanocarriers. Confocal microscopy images showed a well colocalization of the fluorescence of ICG (red) and LysoTracker (green) after incubating of ICG‐PtMGs@HGd with the murine breast cancer cell line 4T1 cells for 1 h , indicating that ICG‐PtMGs@HGd could effectively achieve endosome/lysosome escape after cellular uptake, which is a critical prerequisite for cancer therapy. In contrast, nearly no fluorescence was observed with free ICG after 6 h incubation, which might be attributed to the reduced degradation of ICG after loading into the PtMGs@HGd nanocarriers.The cellular uptake and the endocytosis mechanism of ICG‐PtMGs@HGd were investigated. As it has been widely believed that the cellular endocytosis of the nanoparticles is closely associated with the endolysosomal compartments,39 we −1, PtMGs@HGd: 320 µg mL−1) , indicating that ICG‐PtMGs@HGd did not induce apparent cytotoxicity and had excellent biocompatibility. We then quantitatively elucidated the photoinduced antitumor efficiency of the various preparations. As shown in Figure 1O2 production in PDT effect derived from the insufficient oxygen, but could be significantly improved when adding H2O2. This could be attributed to the elevated continuous O2 supplementation via the catalyze effect of Pt enzyme that was encapsulated in ICG‐PtMGs@HGd. These results were further confirmed by fluorescence costaining using calcein‐AM/PI to visualize the live (green) and the dead (red) cells, respectively. Consistent with the findings in MTS assay, we observed that under normoxia condition, the apoptosis of 4T1 cells was significantly higher when treated with ICG‐PtMGs@HGd than treated with free ICG or PtMGs@HGd  and a cell survival rate of 17.3% was achieved, demonstrating enhanced PDT effect by Pt catalysis‐induced O2 generation. These results together with the in vitro cytotoxicity assay and the live/dead staining assay, demonstrated that ICG‐PtMGs@HGd nanoparticles could effectively kill tumor cells by synergistic O2‐evolving PDT/PTT effect to overcome hypoxic tumor environment. Tumor‐associated fibroblast cells and extracellular matrix in the TME, which contains collagen, glycosaminoglycans, and proteoglycans, have become a primary barrier for diffusion of multifunctional nanoparticles, so the deep penetration ability of the nanomaterials is vitally important for cancer therapy. Here the penetration ability of ICG‐PtMGs@HGd was investigated via 3D spheroids tumor model, which can mimic the pathological penetration barrier of nanoparticles in the solid tumor. The Z‐stack scanning exhibited stronger fluorescence signals in the ICG‐PtMGs@HGd group compared with free ICG  after intravenous injection of the nanoparticles, and found that the fluorescence in the tumor region gradually increased over time in both free ICG or ICG‐PtMGs@HGd injected mice Figure 4a,b. TheSince the high tumor uptake of ICG‐PtMGs@HGd by fluorescence imaging, we expected that these nanocarriers would also have outstanding contrast MSOT imaging owing to their strong capability in NIR absorption and photothermal conversion. We first measured the multispectral optoacoustic intensity of ICG‐PtMGs@HGd at different concentrations of ICG, and found that the MSOT signals increased linearly with ICG concentration , suggesting the ICG‐induced capability of MSOT imaging in ICG‐PtMGs@HGd. We then evaluated the capability of ICG‐PtMGs@HGd for MSOT imaging in vivo by acquiring MSOT and 3D orthogonal MSOT images of the tumors at various time points post intravenous injection. We found that the MSOT signals of ICG‐PtMGs@HGd appeared at the tumor margin 2 h postinjection, and then gradually increased over time and achieved the maximum at 12 h postinjection Figure , demonst The CT contrast of the ICG‐PtMGs@HGd dispersions was investigated by phantom images in vitro at different Au concentrations , a clinically used MRI contrast agent, as the control. As expected, the in vitro T1‐weight MR images showed that the MRI signals from both ICG‐PtMGs@HGd and Gd‐DTPA significantly increased with Gd concentration, and a linear correlation was found between the longitudinal relaxation rate (1/T1) of water protons and the concentration of Gd (III) , confirming the effective MRI performance of Gd (III). The MRI signal from ICG‐PtMGs@HGd was much higher than the one from Gd‐DTPA at each concentration of Gd , indicating better MRI performance of ICG‐PtMGs@HGd compared to Gd‐DTPA. The molar relaxivity (r1), an important parameter that determines the efficiency of an MRI contrast agent, was calculated based on the slope of the line representing the linear correlation between 1/T1 and the concentration of Gd (III). As shown in Figure S4b (Supporting Information), the r1 value of ICG‐PtMGs@HGd nanoparticles was 35.69 mm−1 s−1, 2.2‐fold higher than that of Gd‐DTPA (16.52 mm−1 s−1), further demonstrating the excellent Gd‐mediated MRI capacity of this nanoparticle. A clear outline of the tumor was delineated in the in vivo T1‐weight MR images 12 h postintravenous injection when the brightest image with a sharp border was achieved .We quantitatively investigated the biodistribution of ICG‐PtMGs@HGd in mice by determine the Au and Pt content in the tumors and major organs, including the heart, liver, spleen, lung, and kidney, based on the percentage of injected dose per tissue gram (% ID/g) via ICP‐MS spectrometry Figure . After i48 To 2.7α (HIF‐1α), a protein that has been reported to be upregulated under the hypoxic condition, to track the hypoxia area. Immunofluorescent staining showed that the tumor treated with ICG‐PtMGs@HGd exhibited significant lower content of HIF‐1α compared to the one treated with saline or ICG‐MGs@HGd  exhibited the temperature elevation with increasing irradiation time compared to the negligible temperature elevation in the PBS group Figure , and the2.8 Thus, we also investigated the effect of ICG‐PtMGs@HGd to inhibit tumor metastasis in vitro and in vivo. As shown in Figure 2O2 showed the lowest cell migration, indicating enhanced inhibition of the cell migration by the synergistic phototherapy. The tumors and the major organs of the tumor‐bearing mice after treatment were further collected for H&E and Ki‐67 immunostaining to investigate the extent of tumor metastasis in vivo. H&E staining showed that saline group possessed clear metastatic sites in the lung and the liver, which were characteristic with the infiltrated tumor cells , a row‐normalized dataset was generated with the standard of log2 fold‐change cutoff ≥1 and p‐value ≤0.05 according to the definition, and a hierarchical clustering of genes expressed after the combined phototherapy was calculated. A large number of DEGs were found in the ICG‐PtMGs@HGd + laser group compared to the control group , homo sapiens tumor protein p73 (TP73) and secreted frizzled‐related protein 5, (Sfrp5)) and the genes that negatively regulate cell proliferation , zinc finger and BTB domain containing 16 (Zbtb16)) were obviously upregulated , Ctnna2) that was the structural constituent of cytoskeleton and cadherin binding and Epha1 (recombinant human ephrin A receptor 1) that was a protein‐tyrosine kinase) were also found to be upregulated  ligand 8 (Ccl8), lymphatic vessel endothelial hyaluronan receptor‐1 (Lyve1) and C‐type lectin domain family 4 member E (Clec4e)) that play antitumor immunogenic roles were upregulated and three gene (single copy nuclear gene (Scng), indoleamine 2,3‐dioxygenase 2 (Ido2), and colony stimulating factor 3 (Csf3)) that are related to cell‐mediated immunosuppressive responses were downregulated  were mainly related to the immunosuppressive response for activation and proliferation of the main effector cells  in cell‐mediated immunity. Overall, the six changed immune‐related DEGs showed that the synergistic phototherapy indeed gave rise to the immunological responses, which were also reported in the other PDT/PTT synergistic phototherapies. PDT has been demonstrated to induce immunogenic cell death (ICD) and activate an adaptive immune response against tumor‐associated antigens. As PDT is a typical oxygen‐consuming process whose effect can be severely limited by tumor hypoxia, thus the oxygen supplied nanoparticles can elicited ICD upon abundant ROS in PDT. Similarly, PTT has been shown to trigger the thermal ablation‐induced tumor ICD and cause the release of tumor antigens and endogenous adjuvants  to stimulate the immune responses. Our ICG‐PtMGs@HGd nanoparticles provided an O2 self‐enriched nanoplatform for enhanced PDT/PTT, and might consequently enhance the synergistic phototherapy‐induced antitumor immune responses as the ICD caused by the phototherapy may trigger the maturation of dendritic cells and subsequently activated specific effector cells  to further inhibit the tumor growth and metastasis. However, more efforts would be put on the functional studies to further elucidate the synergistic phototherapy‐mediated immune responses.To identify interrelated altered genes related to the 4T1 breast cancer in response to the synergistic phototherapies, the correlated genes were analyzed from the candidates cancer gene database.55 Apod Figure , suggestd Figure , indicatd Figure , suggest2.10Figure  These results, together with the H&E staining of the main organs after the treatment with ICG‐PtMGs@HGd, that showed on noticeable organ impairment nor obvious inflammation or necrosis  was purchased from Innochem Co., Ltd. 2‐aminoterephthalic acid (NH2‐BDC) and 11‐mercaptoundecanoic acid (MUA) were brought from Aladdin. Dimethyl formamide (DMF) and acetic acid were obtained from Beijing Chemical Works. Chloroplatinic acid (H2PtCl6·6H2O), chloroauric acid (HAuCl4), N‐(3‐(dimethylamino)‐propyl)‐3‐ethylcarbodiimide hydrochloride (EDC), indocyanine green (ICG), N‐hydroxysuccinimide (NHS), HSA, and Gd(NO3)3·6H2O were all obtained from J&K Scientific Co., Ltd. Potassium carbonate was obtained from VETEC (Sigma‐Aldrich). Formaldehyde was brought from Xilong Scientific Co., Ltd. 1,3‐Diphenylisobenzofuran (DPBF) and Ti(SO4)2 were obtained from Energy Chemical (Shanghai). PVP (Mw = 58 000), PVP (Mw = 40 000), 4′, 6‐diamidino‐2‐phenylindoles (DAPI), MTS cell proliferation colorimetric assay kit, calcein‐AM/PI double stained kit, annexin V‐FITC/PI apoptosis detection kit and DCFH‐DA were all obtained from Solarbio. Penicillin/streptomycin and Lyso‐Tracker‐Green DND‐26 were bought from Invitrogen (USA). Fetal bovine serum (FBS) and RPMI Media 1640 were purchased from Gibco Co., Ltd. HIF‐1α (Alexa Fluor 488 Conjugate) and phalloidin (Rhodamine Conjujugate) were bought from Abcam.Zirconium chloride (ZrCl2) generation was determined with portable dissolved oxygen meter . The concentration of Au was determined by inductively coupled plasma mass spectroscopy (GGL‐ICP‐MS) . The laser of 808 nm was administrated by power‐tunable infrared laser . Thermal images were captured by using a DT‐980 infrared camera  and analyzed by infrared image software.Topography and elemental mapping images were recorded with Ht‐7700 transmission electron microscopy  and high resolution TEM (HRTEM) , respectively. UV–vis spectra were captured by Lambda UV–vis spectrophotometer . Dynamic light scattering (DLS) for particle size and zeta potential were determined by Zetasizer Nano ZS . The XRD patterns were tested on a TZY‐XRD . The experiment of Barrett‐Joyner‐Halenda model from adsorption branch of isotherms was performed with automatic surface area and porosity analyzer . The circular dichroism (CD) spectra were measured with circular dichroism chiroptical spectrometer spectropolarimeter system . Oxygen (O2PtCl6⋅6H2O (0.196 mmol) and 444 mg of poly(vinylpyrrolidone)  were dissolved in 50 mL of ethylene glycol in a two‐neck round‐bottomed flask. The solution was reacted in an oil bath at 180 °C for 10 min, and the obtained PVP‐protected Pt nanoparticles were then precipitated in acetone after cooling down. Subsequently, the Pt nanoparticles were centrifugated , cleaned with acetone and hexane to remove excess free PVP, and redispersed in DMF (20 mL) (with Pt nanoparticles concentration of about 1 mg mL−1) for further use.PtMGs and MGs were fabricated first. Typically, H Briefly, ZrCl4 (8.75 × 10−3m) and NH2–BDC (8.01 × 10−3m) were dissolved in DMF (5 mL) respectively and were subsequently mixed in the reactor followed by adding acetic acid (1.2 mL) and above Pt nanoparticles (2 mL). The whole mixture was sealed into the vacuum drying oven to crystallize at 120 °C for 12 h. After that, the obtained product was collected, washed by centrifugation with DMF for three times, then transferred into ethanol at 37 °C for 12 h and last dispersed in 5 mL of deionized water for following use. For preparation of MOFs without Pt decoration, we further added the same amount of ZrCl4, NH2–BDC and acetic acid (except for Pt nanoparticles), and all the other steps are the same.The Pt‐decorated MOF  nanoparticles were prepared based on the previous method with minor modification.26 Bri2CO3 (50 mg) and HAuCl4  were mixed in deionized water (100 mL) under stirring at 900 rpm for 10 min in the dark. Next, 1 mL of obtained PtMs solution was added into the gold growth solution followed by adding 10 mL of 1 mg mL−1 PVP (Mw = 40 000) and formaldehyde (2 mL). The mixture was then reacted for 2.5 min under stirring of 450 rpm. Last, the obtained PtMGs nanoparticles were collected and subsequently purified with water by centrifugation  for several times. The MGs nanoparticles were prepared using MOFs as template, and all the other steps are the same.The gold growth solution was first synthesized as follows: K50 × 10−3m, 1.5 mL Specifically, HSA (0.25 g) was dissolved in deionized water (9 mL) at 37 °C followed by adding of Gd(NO3)3  aqueous solution for 5 min's reaction. Subsequently, NaOH solution  was rapidly introduced and stirred at 37 °C overnight. Last, HGd mixture was purified by dialysis method for 24 h.The PtMGs‐COOH nanoparticles were then constructed. The PtMGs were scattered ethanol solution with ultrasound. After that, 11‐mercaptoundecanoic acid (MUA) with 10 times’ weight of PtMGs, was added for reaction of 12 h to get the PtMGs‐COOH nanoparticles. Last, the obtained nanoparticles were collected and purified with ethanol by centrifugation . After that, the HGd hybrids was synthesized according to the reported method.65 Spe−1, 20 mL) for activating the carboxyl group. About 2 h later, the redundant compounds were discarded by centrifugation, and HGd hybrids (0.35 mL) was introduced for another 24 hours’ reaction. The obtained PtMGs@HGd were collected and further purified by centrifugation.Finally, PtMGs@HGd nanoparticles were prepared. Specifically, EDC (3.2 mg) and NHS (2.4 mg) were injected into PtMGs‐COOH solution  and then incubated for 24 h at room temperature. After that, the complex was washed with centrifugation for several times to remove unloaded ICG, then the supernatant and all the washing solution were collected to accept the absorbance measure at 780 nm for evaluating the loading capacity of ICG based on the following formula:MICG, MuICG, and MICG‐PtMGs@HGd were the total mass of ICG, unloaded ICG and ICG‐PtMGs@HGd, respectively.PtMGs@HGd (0.4 mg) were separately added into 1 mL ICG solution . ICG‐PtMGs@HGd (8 mL) was put in a dialysis bag and then immersed into 60 mL PBS receptor fluid. For each predesigned time interval, 2 mL of the PBS solution was taken out and tested by UV–vis spectrum at the wavelength of 780 nm to quantify the amount of released ICG. After that, 2 mL of new PBS solution was further introduced to maintain consistence. All the release studies were performed in a dark environment.2O2 consumptions were first investigated. The titanium sulfate (Ti(SO4)2) were used as an H2O2 indicator, whose color changes from colorless to yellow when specifically react with H2O2. The catalytic effect of ICG‐PtMGs@HGd was evaluated by mixing H2O2 (1 × 10−3m) and ICG‐PtMGs@HGd (80 µg mL−1) in PBS (solution A) shaken at 37 °C. Ti(SO4)2 (50 mg) was dissolved into of H2SO4 (8.33 mL) and further diluted with deionized water to 50 mL volume (solution B). For every 30 min, 1 mL of solution A was centrifuged, then 0.35 mL of the supernatant was mix with 2.65 mL of solution B and its UV–vis absorbance at 405 nm was measured. The PBS and ICG‐MGs@HGd groups were also performed based on the former methods as control. Continuous catalytic behavior was tested by introducing the H2O2 to the ICG‐PtMGs@HGd for several repeated times, and the remaining concentration of H2O2 was detected with the same method.The Hith H2O2.33 The2O2, ICG‐PtMGs@HGd was ultrasonically scattered in water with 1 × 10−3m H2O2, then the oxygen content was measured by a portable dissolved oxygen meter over time. In addition, the successive O2 production by introduce of H2O2 (1 × 10−3m) into ICG‐PtMGs@HGd suspension every 30 min was also measured over time.To investigate the capability of oxygen generation with the intracellular H−1, respectively) and 0.5 mL different formulations of PBS, HGd, PtMGs@HGd, ICG, and ICG‐PtMGs@HGd  were put into the eppendorf tubes, then exposed to laser irradiation . The real time thermal images and the corresponding temperature quantitative value were determined by an infrared thermal camera. The photothermal stability of ICG‐PtMGs@HGd was performed with cyclical laser irradiation  for 420 s, then naturally cooled without irradiation for 600 s. This cycle was then repeated for four times. The photothermal conversion efficiency (η) was calculated based on the reported method. Briefly, ICG‐PtMGs@HGd  was irradiated with a NIR laser  until the formulation reached a stable value, then the nanoparticle was naturally cooled to room temperature. The temperature changes during the whole process were recorded and the η value was calculated (Supporting information). To test whether photothermal effect can enhance the Pt catalyzed O2 generation, the oxygen generation of the PtMGs@HGd nanoparticles were measured with or without laser irradiation  using the dissolved oxygen meter under the introduction of H2O2.To explore the photothermal performance, PtMGs@HGd solution (0.5 mL) of various concentrations  was mixed with 2 mL different formulations ; ICG‐PtMGs@HGd, ICG‐PtMGs@HGd + H2O2 and ICG‐PtMGs@HGd + H2O2 (hypoxia conditions) with the same amount of ICG (4 µg mL−1) and PtMGs@HGd (32 µg mL−1). The solutions were further exposed to NIR laser for 10 min, then ultraviolet absorption intensity of DPBF at 410 nm was recorded. Herein, hypoxic condition was achieved by blowing 1% oxygen into the solution for 30 min before the test.1,3‐Diphenylisobenzofuran (DPBF) was chosen to test the production of singlet oxygen by ICG‐PtMGs@HGd in the presence of H2O2.34 Bri2.4T1 cells were purchased from National Infrastructure of Cell Resource (Beijing) and cultured in RPMI‐1640 culture medium with 10% FBS and 1% penicillin‐streptomycin at 37 °C in a humidified incubator containing 5% CO5 cells per well), then ICG and ICG‐PtMGs@HGd (with the same ICG concentration of 20 µg mL−1) were added for incubation of 1 and 6 h, respectively. The 4T1 cells were washed with PBS, sequentially stained with Lyso‐Tracker‐Green DND‐26 and DAPI, and further captured with confocal microscopy . The Pearson's correlation coefficient was obtained based on the Image J software.For cellular uptake studies, 4T1 tumor cells were planted into a CLSM dishes  for 24 h. Then the culture medium was eliminated and 20 µL of MTS solution was introduced into each well and incubated for another 2 h. Finally, the absorbance at 490 nm of was measured using a microplate reader . For exploring the cytotoxicity of the enhanced PDT/PTT effect in the normoxic and hypoxic tumor microenvironment, 4T1 cells were added with different formulations of PBS, PtMG@HGd, ICG, ICG‐PtMG@HGd, and ICG‐PtMG@HGd + H2O2  for 6 h under normoxic and hypoxia condition . Next the cells of different groups were irradiated with an NIR laser , then incubated for another 18 h and determined by MTS assay. The PBS group without laser irradiation was employed as control.MTS experiment was first applied to test the cytotoxicity of the obtained ICG‐PtMGs@HGd formulations. 4T1 were preseeded into a 96‐well plate at the density of 5 × 10ctively).66 Nex6 cells per well), then added with different preparations of PBS, PtMG@HGd, free ICG, ICG‐PtMG@HGd, and ICG‐PtMG@HGd + H2O2  for 6 h under normoxic and hypoxia condition  before 5 min's laser irradiation. Subsequently, the tumor cells were washed with PBS, incubated with AM/PI kit and then observed by confocal fluorescence microscopy . The PBS group without laser irradiation was used as control.To observe the therapeutic effect vividly, the synergistic PDT/PTT effects of ICG‐PtMGs@HGd in hypoxic tumor microenvironment were further verified by AM/PI cell costaining assays. Specifically, 4T1 cells were preseeded into confocal dishes  for overnight under hypoxia conditions (1% oxygen concentration) before NIR laser. Afterwards, 4T1 cells were collected, stained with annexin V/PI kit, and then detected by flow cytometer . The PBS group without laser irradiation was used as control.To determine the apoptotic and necrotic cells, the 4T1 cells were preseeded into a six‐well plate for 24 h at a density of 1 × 102O2  for 4 h, and irradiated with 2 min's NIR laser . Then the old medium was removed 24 later, and replaced with FBS‐free medium for another 6 h’ incubation. In addition, 4T1 cells were washed, digested, and put into the upper chambers of a transwell with FBS‐free medium (100 µL), then the culture medium containing FBS was added into the lower chambers. Last, 4T1 cells in the upper chambers were wiped off and washed after 24 h, then the membrane was stained with 0.1% crystal violet and observed by a microscope for migrated cells.The inhibition effect of tumor metastasis was tested by cell migration assay according to a transwell model. Briefly, 4T1 cells were fed with PBS, free ICG, PtMGs@HGd, ICG‐PtMGs@HGd, and ICG‐PtMGs@HGd + H2O2 assay, was used to evaluate the intracellular H2O2 concentration. Typically, 4T1 cells were seeded on the chambered cover glass, and then incubated with PBS or ICG‐PtMGs@HGd (45 and 90 µg mL−1) under normal or hypoxic condition (21% and 1% oxygen concentration) for 24 h and washed with PBS for several times. After that 100 µL DMEM medium containing 100 × 10−6m H2O2 was added and incubated for 1 h. In addition, the cell medium was discarded and washed followed by incubation with MAK164 working solution for another 1 h. After washing with PBS, the intracellular H2O2 concentration was evaluated by fluorescent microscope (ex/em = 490/520).MAK164 (Sigma Aldrich), an intracellular Hntration.21 Typ2 generation, 4T1 cells were seeded on the chambered cover glass. Then, the cells in each group were incubated with PBS or ICG‐PtMGs@HGd under normal or hypoxic condition (21% and 1% oxygen concentration) for 24 h. These cells were reacted with [Ru(dpp)3]Cl2 (10 µg mL−1) (Sigma‐Aldrich) for another 12 h followed by rinsing with PBS to remove free [Ru(dpp)3]Cl2 and the residual particles. The intracellular oxygen level was detected based on the fluorescence of [Ru(dpp)3]2+ (ex/em = 450/610 nm).For detecting the intracellular Oarticles.35 The−1). 4T1 cells were fixed with paraformaldehyde, permeabilized with PBS containing 0.2% Triton X‐100 and then blocked with PBS containing 0.05% Tween‐20 buffer and 1% bovine serum albumin. After that, the tumor cells were incubated with anti‐HIF‐1α antibody  at 4 °C overnight, washed with PBS containing 0.2% Triton X‐100 and 2% Tween‐20 buffer for three times, and incubated with CoraLite488‐conjated AffiniPure Goat Anti‐Rabbit LgG  for 1 h at room temperature. Finally, the cells were further washed with PBS containing 0.2% Triton X‐100 and 2% Tween‐20 buffer for three times, incubated with rhodamine conjugate‐phalloidin  at room temperature for 1 h and then observed by CLSM.The cellular hypoxia condition under normoxic or hypoxic environments was evaluated following treated with PBS or ICG‐PtMGs@HGd , and further fed with different preparations  at 37 °C under the normoxia or hypoxia condition (21% and 1% oxygen concentration). After incubation for 4 h, the cells were washed with PBS, exposed to NIR laser  for 3 min, incubated with DCFH‐DA (10 × 10−6m) for 30 min, then observed by CLSM.DCFH‐DA was selected as a typical ROS indicator to detect intracellular ROS of ICG‐PtMGs@HGd in 4T1 cells.37 Bri Typically, 50 µL of 1% (w/v) agarose solution was introduced into a 96‐well plate and exposed to UV irradiation for 30 min. Then 1000 cells (200 µL) cell was added and further incubated for one week to form the 4T1 MCSs. To investigate the penetrability, the tumor spheroids were fed with free ICG (20 µg mL−1) or ICG‐PtMGs@HGd . After incubation for 24 h, the spheroids were put into CLSM culture dishes, washed with PBS and observed with CLSM with Z‐stack scanning.Multicellular cancer spheroids (MCSs) was constructed to perform an intermediary between the 2D monolayer cell model and the solid tumor model.67 TypIn vivo tumor animal model was obtained by subcutaneously inoculating ∼2 × 106 4T1 cells into the right flank part. When the tumor volume reached about 150 mm3, the animal experiments could be carried out in accordance with the guidelines approved by the Ethics Committee of the National Center for Nanoscience and Technology of China.The female nude mice and BALB/c mice (6–8 weeks old) were bought from Charles River . −1 of ICG) by intravenous injection. After that ICG fluorescence images were acquired at the predesigned time points by IVIS Spectrum imaging system . At the time point of 24 h, mice were sacrificed, then the major organs and tumors were collected for ex vivo fluorescence imaging and quantitative biodistribution analysis.In order to investigate the fluorescence imaging, the tumor cells‐bearing mice were administrated with free ICG and ICG‐PtMGs@HGd (1.0 mg kg−1) by the InVision 128 MSOT system . In order to test the in vivo multispectral optoacoustic imaging, 200 µL of ICG‐PtMGs@HGd (1.0 mg kg−1 of ICG) was injected into the tumor‐bearing mice intravenously, then the mice were anesthetized and scanned at predesigned time points  by MSOT system.The multispectral optoacoustic intensity of ICG‐PtMGs@HGd in vitro was first measured at various ICG concentrations  with X‐ray CT . To obtain the in vivo CT imaging, the ICG‐PtMGs@HGd (20 mg kg−1) was administrated into the tumor sites and the CT images were recorded after 12 h injection.The hounsfield units (HU) signal of ICG‐PtMGs@HGd in vitro was first carried out at various Au content  by 7.0 T small animal MRI instrument . To test in vivo MR images, the tumor‐bearing mice were injected with ICG‐PtMGs@HGd (0.05 mmol Gd kg−1) by intravenous injection, then the MR images were acquired at predesigned time points .The in vitro T−1)‐injected mice were solubilized with aqua regia to get Au and Pt content by ICP‐MS test after 24 h injection. To test the blood circulation, the blood samples (200 µL) were collected at specific time points, weighted, and then solubilized with nitrohydrochloric acid. Last, the Au contents of the different samples were investigated by ICP tests to obtain the bloodstream pharmacokinetics profile.In order to investigate the biodistribution of ICG‐PtMGs@HGd, tumors and major organs  of ICG‐PtMGs@HGd (9 mg kg−1) by intravenous injection. About 24 h later, mice were sacrificed, then tumors were excised, cut into proper slices, incubated with anti‐CD31 antibody  and DAPI, and further observed on a LSCM.To study the diffusion of ICG‐PtMGs@HGd under tumor microenvironment, the tumor bearing mice were treated with free ICG and ICG‐PtMGs@HGd   and DAPI, and further observed under a LSCM.For investigating the intratumoral hypoxia, tumor bearing mice were intravenously injected with PBS, ICG‐MGs@HGd and ICG‐PtMGs@HGd, respectively. About 24 h later, the mice were sacrificed, then the tumors were excised, cut into slices, stained with anti‐HIF‐1 alpha (HIF‐1−2) was applied at the tumor parts of the tumor‐bearing mice after intravenous injection of PBS, ICG, PtMGs@HGd, ICG‐MGs@HGd, and ICG‐PtMGs@HGd  for 12 h. The thermographic images of the tumor sites were recorded by an infrared thermal camera to exhibit the temperature changes.In order to investigate in vivo photothermal effect, NIR laser  saline; b) saline + laser; c) PtMGs@HGd + Laser; d) Free ICG + laser; e) ICG‐MGs@HGd + Laser; f) ICG‐PtMGs@HGd + Laser. Each treatment with the same amount of ICG (1 mg kg−1) and Au  was injected from tail‐vein, and the photothermal therapy  was performed for 5 min after12 h injection. Tumor size and body weight of mice were measured every other day after irradiation. The tumor volume of the mice could be calculated according to the reported formula (volume = width2 × length/2). On the day of 18, mice of each group were sacrificed and the major organs as well as the tumors were harvested. The obtained tumors were weighted, imaged, and further processed forhematoxylin/eosin (H&E) staining and TUNEL staining. As a separate experiment, the survival rate of the mice which were treated under the same conditions was tested. To evaluate the inhibited effect of tumor metastasis, the obtained organs were disposed with H&E and ki67 staining to investigate the extent of tumor metastasis.After the tumor volume reached ≈150 mmength/2).68 On The tumors of saline and ICG‐PtMGs@HGd + Laser groups were stripped. The responding total RNA was extracted for the subsequent RNA sequencing (RNASeq) test. All the RNA‐Seq reads were mapped and the human genome sequence  were used as a reference. The differentially expressed genes (DEGs) were investigated by the softwares HTSeq and DEseq2. The transcript counts and the relative abundance of gene expression were calculated based on the software Cufflinks (version 2.1.1).−1, PtMGs@HGd: 8 mg kg−1). Then 1 mL blood from each mouse was collected for the blood biochemistry analysis 6, 14, and 18 d postinjection. Liver function was tested by measuring serum levels of ALP , AST (aspartate transaminase), and ALT (glutamate transaminase). Kidney function was evaluated by determining BUN (blood urea nitrogen), Cr (creatinine), CAT , and GSH‐PX (glutathione peroxidase) content. Other parameters such as GLU (glucose), MCV (mean corpusular volume), TC , and CHOL (cholesterol) were also tested to further testify the long‐term toxicity of ICG‐PtMGs@HGd.To evaluate the long‐term toxicity of the obtained formulation, healthy Balb/c mice were intravenously administrated with saline and ICG‐PtMGs@HGd (ICG: 1 mg kgP < 0.05 was identified to be statistically significant.Quantitative data were exhibited as means ± standard deviation and performed by ANOVA. Differences among multiple groups were analyzed by one‐way ANOVA. Student‐ Newman‐Keuls test was utilized as post hoc test. The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


GSK3β, through intravitreal administration, abrogated the oxidative stress-derived mitochondrial defect and synaptic neurodegeneration in diabetic RGCs. By contrast, ablation of β-catenin by si-β-catenin abolished the protective effect of GSK3β inhibition on diabetic RGCs by suppression of antioxidant scavengers and augmentation of oxidative stress-driven mitochondrial lesion. Thus, our data identify β-catenin as a part of an endogenous protective system in diabetic RGCs and a promising target to develop intervention strategies that protect RGCs from neurodegeneration at early onset of diabetic retinopathy.Synaptic neurodegeneration of retinal ganglion cells (RGCs) is the earliest event in the pathogenesis of diabetic retinopathy. Our previous study proposed that impairment of mitochondrial trafficking by hyperphosphorylated tau is a potential contributor to RGCs synapse degeneration. However, other molecular mechanisms underlying mitochondrial defect in diabetic retinal neurodegeneration remain to be elucidated. Here, using a high-fat diet (HFD)-induced diabetic mouse model, we showed for the first time that downregulation of active β-catenin due to abnormal GSK3β activation caused synaptic neurodegeneration of RGCs by inhibiting ROS scavenging enzymes, thus triggering oxidative stress-driven mitochondrial impairment in HFD-induced diabetes. Rescue of β-catenin via ectopic expression of β-catenin with a recombinant adenoviral vector, or via GSK3β inhibition by a targeted si- Diabetic retinopathy (DR) remains a leading cause of vision loss in adults world-wide and has long been classified as a microvascular complication of diabetes . HoweverMitochondrial dysfunction is considered as the prime factor responsible for degeneration and loss of neurons in the pathogenesis of neurodegenerative diseases . MaintenMitochondrion is a major site of reactive oxygen species (ROS) production, and also a target for destructive effect of oxidants, indicating the presence of a vicious cycle of oxidative damage , 11. Recin vitro and in vivo [The canonical Wnt/β-catenin signaling has been implicated in the maintenance of development and normal function of specific retinal neurons  as well  in vivo –20. Phos in vivo , 22. In  in vivo  leads to in vivo . Of inte in vivo –28. Loss in vivo , indicatThe aim of the present study was firstly to explore the putative roles of oxidative stress-derived mitochondria damage in diabetic RGCs synapse neurodegeneration in a well-established high-fat diet (HFD)-induced diabetic mouse model, where degeneration of retinal neural cells precedes the development of visible retinal microvascular lesions as we and others reported –33. In aOur previous study showed that HFD for 20 weeks can elicit diabetic RGCs dysfunction and synapse degeneration in the absence of detectable retinal microvascular abnormality , 33. TheCat, Sod1, Sod2, Sod3, Gpx1, Gpx2, Trxr1, and Trxr2 , a generic ROS scavenger, was injected intravitreally into the right eye of HFD-induced diabetic mice, while PBS was injected into the contralateral left eye as a control . Forty-eGiven that transcription activator β-catenin has been reported to regulate the expression of antioxidant scavengers in response to ROS , 27, 28,GSK3β) was intravitreally injected in mice at 20 weeks after HFD, while scramble si-sc was injected in the contralateral eye as a control. Remarkably, knock-down of GSK3β in diabetic retinae  was intravitreally co-administrated with si-GSK3β into the right eye of HFD-induced diabetic mice, while scramble si-sc was co-injected with si-GSK3β in the contralateral eye as a control. As relative to scramble control, knock-down of β-catenin , cytosolic and nuclear levels of active β-catenin, and transcriptional activity of β-catenin in RGCs upon glucolipotoxicity  was purchased from Santa Cruz Biotechnology ; siRNA against GSK3β (si-GSK3β) 5’-CCACTCAAGAACTGTCAAGTA-3’ (sense strand) and scramble control (si-sc) 5’-UUCUCCGAACGUGUCACGUTT-3’ (sense strand) were ordered from Genemall  [TM. Primary antibodies against non-phospho (active) β-catenin at Ser33/37/Thr41 (4270), phospho β-catenin at Ser33/37/Thr41 (9561), total β-catenin (9562), total Akt (4691), phosphorylated Akt at S473 (4060), total GSK3β (9315), phosphorylated GSK3β at S9 (9336), and GAPDH (2118) were ordered from Cell Signalling Technology . Antibodies against synaptophysin (ab32127), Thy1 (ab225), Tuj1 (ab18207), MAP2 (ab75713), PTP1B (ab189179), and histone H3 (ab1791) were purchased from Abcam . Primary mouse anti Flag tag antibody (AE005) was from ABclonal . Secondary antibodies, including Alexa-Fluor-488 goat anti-rabbit, -594 goat anti-mouse, and -405 goat anti-chicken IgG were from InvitrogenTM . Normal donkey serum (ab7475) was purchased from Abcam .A recombinant adenovirus expressing β-catenin with N-terminal Flag tag (Ad-β-catenin) and an empty control (Ad-Ctrl) were purchased from Vigene Bioscience . Short interfering RNA (siRNA) against β-catenin  and their scramble controls  . N-acetyMale C57BL/6 mice were purchased from Guangdong Medical Laboratory Animal Center . Starting at 5 weeks of age, mice were fed either a HFD  or regular standard laboratory chow (RD) as controls  as we previously described  for 20 wGlucose or insulin tolerance tests were performed by an oral administration of D-glucose (1 g/kg) (OGTT) or an intraperitoneal injection of insulin  (IPITT) after an 8 h fast, respectively, as previously described . Blood g2 for 2 min to one eye from a RETI-Port/Scan 21 recorder ,VEP was performed as described in our previous studies , 33. In Retinal vascular permeability was determined using Evans Blue (EB) as tracer. Briefly, mice were anesthetized and received a tail vein injection of EB dye . Two hours later, a 0.2 ml of blood sample was collected from mice which were then perfused via the left ventricle with 1 ml warm PBS followed by 1% paraformaldehyde. The eyes were enucleated and retinae were dissected. For retinal EB angiography, retinae were flat-mounted and imaged at 620 nm with a Leica DMI4000B inverted fluorescence microscope . Alternatively, retinae and blood samples were treated with and without dimethylformamide , respectively. Extract supernatants were measured by a spectrophotometer at 620 nm and 740 nm, respectively. Retinal vascular permeability was expressed as microgram of EB per milligram of retinal proteins after normalization by concentrations of total retinal protein as previously described .SEM was performed as described in our previous studies , 33. In TEM was performed as previously described . BrieflyRetinae and primary RGCs immunofluorescence analysis was performed as previously described , 33. In 7 cells/dish were fractionated using the FractionPREP Cell Fractionation Kit . Equal amounts of protein resolved by SDS-PAGE were immunoblotted as previously described [Whole retinae were lysed by sonication in 150 mM NaCl, 50 mM Tris (pH 7.4), 1 mM EDTA, 1% NP-40, 0.5% Triton X-100, and 2 mM NaVO3, in the presence of protease and phosphatase inhibitors. For detection of protein expression of β-catenin in the cytosol and nucleus, primary RGCs at a density of 1 x 10escribed  using prThe activity of complex I-IV in mitochondrial electron respiratory chain was determined with the Micro Mitochondrial Respiratory Chain Complex I, II, III or IV Activity Assay Kit  according to the manufacturer’s instructions. Briefly, two retinae freshly isolated from two respective mice in the same group were pooled and suspended in the mitochondrial complex extraction buffer, followed by gentle homogenization. The homogenate was centrifuged at 600 g for 10 minutes at 4 °C to remove cell debris and nuclei, and the supernatant was centrifuged again at 11,000 g for 15 minutes at 4 °C. The resultant pellet was resuspended in the extraction buffer and crushed by ultrasonication. Protein concentration of the extracted complex was determined by the bicinchoninic acid (BCA) assay . The complex activity of mitochondrial homogenates in the respective reaction buffer was then measured spectrophotometrically at 340 nm (complex I), 605 nm (complex II), and 550 nm (complex III and IV), respectively. Mitochondrial complex activity was expressed as nmol/min/mg protein. Three independent experiments were performed in duplicate for each group.In brief, freshly isolated retinae were homogenated and centrifuged at 1000 g for 20 min at 4 °C. The supernatant was quantitatively assessed using a BCA assay kit. The activity of catalase (CAT), total superoxide dismutase (tSOD), manganese superoxide dismutase (MnSOD), Copper/Zinc superoxide dismutase (CuZn-SOD), and glutathione peroxidase (GPx) was measured with the respective commercial assay kit  according to the the manufacturer’s instructions. Enzyme activity was normalized by total retinal protein concentrations and expressed as unit/mg protein.Briefly, retinae or primary RGCs were homogenated and centrifuged at 1000 g for 20 min at 4 °C. Protein concentration in the supernatant was quantified by BCA assay. Contents of 4-HNE were determined with a 4 - hydroxy nonene aldehyde (4-HNE) ELISA assay kit from j&l Biological Industrial Co., Ltd. , normalized by total retinal protein concentrations, and expressed as nmol/mg protein.The RNA from retinae was extracted with the RNeasy Mini Kit . Reverse transcription was carried out using Random hexamers and SuperScript-III (Invitrogen). Quantitative real-time PCR was performed with the Applied Biosystems 7300 real-time systems using real-time PCR Master Mix (SYBR Green). The primer sequences are listed in Additional file 2 .7 pfu/eye) was injected intravitreally into the right eye of HFD-fed mice, while an empty vector Ad-Ctrl was injected into the contralateral left eye as a control. For GSK3β depletion, si-GSK3β (2 μg/μl) was administrated into one eye and si-sc was applied into the contralateral eye. For co-repression of GSK3β and β-catenin, si-GSK3β (2 μg/μl) mixed with si-β-catenin (100 nM) or control si-sc was intravitreally injected into the right eye or contralateral left eye of HFD-fed mice, respectively.Intravitreal injections were performed as previously described , 55. In β-catenin or si-sc with a Micropoly-transfecterTM cell reagent  according to the manufacturer’s instructions. Twenty-four hours after transfection, cells were subjected to conditioned medium in the presence of TWS119 for another 24 h.Primary RGCs were isolated from neonatal (1-3 days old) Sprague-Dawley rats as we previously described . Cells aIn brief, primary RGCs treated with different conditions were incubated with 10 μM DCFH-DA (Sigma-Aldrich), an indicator of general oxidative stress, for 1 h at 37 °C under 5% CO2. Cell suspensions were then centrifuged at 800 g for 5 min and washed with PBS twice. The fluorescence intensity was determined by a flow cytometer  at excitation/emission of 485/528 nm, respectively. Three independent experiments were performed in duplicate for each condition.The mitochondrial membrane potential (MMP) was determined with a fluorescent indicator JC-1 . Briefly, primary RGCs upon different treatment were incubated with 200 μM JC-1 for 20 min at 37 °C. JC-1 can enter the mitochondrial matrix to form polymer and generate red fluorescence. At low MMP, JC-1 exists in the form of monomer in the cytoplasm and yields green fluorescence. After washing twice with PBS to remove the remaining reagents, images were obtained with an OLYMPUS FV1000 confocal microscope. The immunufluorescence intensity was determined per cell in four consecutive fields that were randomly selected by Image-Pro Plus software. The ratio of red to green fluorescence intensity reflected the level of the MMP.TM cell reagent. Twenty-four hours after transfection, cells were subjected to the respective treatment for another 24 h. Cells were harvested and analyzed by the dual-luciferase assay kit . Three independent experiments were performed in duplicate for each condition.TOPFlash reporter or FOPFlash reporter  was co-transfected with an internal control Renilla luciferase reporter pRL-TK  into primary RGCs with the Micropoly-transfectert-test was used to analyze statistical significance between two groups. Statistically significant was considered when P<0.05.Data were expressed as mean ± SEM. All statistical analyses were performed with GraphPad Prism 8.0 . Student’s Animals were handled in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [(NIH) publication no. 85–23, revised 1996). All the animal experiments and maintenance were approved by the Laboratory Animal Ethics Committee of Shenzhen University (Permit Number: 201412042).Supplementary FiguresSupplementary Table 1


Cartilage endplate (CEP) degeneration has been considered as one of important factors related to intervertebral disc degeneration (IVDD). Previous researches have showed that Rac1 played a pivotal role in chondrocyte differentiation. However, the effect of Rac1 during the process of CEP degeneration remains unclear. Herein, we explored the effect of Rac1 on CEP degeneration and elucidated the underlying molecular mechanism. We found expression of Rac1‐GTP increased in human‐degenerated CEP tissue and IL‐1β‐stimulated rat endplate chondrocytes (EPCs). Our study revealed that Rac1 inhibitor NSC23766 treatment promoted the expression of collagen II, aggrecan and Sox‐9, and decreased the expression of ADTAMTS5 and MMP13 in IL‐1β‐stimulated rat EPCs. Moreover, we also found that NSC23766 could suppress the activation of Wnt/β‐catenin pathway, suggesting that the beneficial effects of Rac1 inhibition in EPCs are mediated through the Wnt/β‐catenin signalling. Besides, puncture‐induced rats models showed that NSC23766 played a protective role on CEP and disc degeneration. Collectively, these findings demonstrated that Rac1 inhibition delayed the EPCs degeneration and its potential mechanism may be associated with Wnt/β‐catenin pathway regulation, which may help us better understand the association between Rac1 and CEP degeneration and provide a promising strategy for delaying the progression of IVDD. Human CEP tissue collection and experiments that involved human CEP were approved by the Second Affiliated Hospital and Yuying Children's Hospital of Wenzhou Medical University Ethics Committee (L‐2018‐46) and followed the guidelines of the Helsinki Declaration.2.2Recombinant rat IL‐1β was obtained from Peprotech. NSC23766 was obtained from MedChemExpress. XAV‐939 and SKL2001 were purchased from Meilunbio. Antibodies against Rac1‐GTP, Rac1‐Total and Sox‐9 were purchased from Cell Signaling Technology. Antibodies against β‐actin and normal rabbit IgG were purchased from Santa Cruz Biotechnology. Antibodies against aggrecan, collagen II, ADAMTS5 and MMP13 were purchased from Abcam.2.33 and treated with 0.2% type II collagenase for 4 hours at 37℃. After washing with phosphate‐buffered saline (PBS), human EPCs were added to the 10‐cm culture plates with appropriate density, and cells were cultured in DMEM/ F12 supplemented with 10% foetal bovine serum (FBS) and antibiotics (1% streptomycin/penicillin) incubate in 5% CO2 and 37°C. The complete medium was changed every other day, and the first three passages of chondrocytes were used for the experiments.The normal human CEP tissue  was obtained from patients undergoing treatment of lumbar fracture with lumbar internal fixation. The degenerated human CEP tissues  from IVDD patients. To isolated primary human endplate chondrocytes (EPCs), CEP tissue was cut up into 1 mm2.42 and 37°C. The complete medium was changed every other day, and the first three passages of chondrocytes were used for the experiments.Cartilaginous endplate tissues were extracted from the tails of 2‐week‐old rats  under a dissecting microscope. The cartilage tissues were digested with 0.2% type II collagenase for 4 hours at 37°C. Next, the rat EPCs were added to the 10‐cm culture plates with appropriate density and cultured in DMEM/F12 supplemented with 10% FBS and antibiotics (1% streptomycin/penicillin) and maintained in 5% CO2.5w/v) sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and were transferred to a polyvinylidene difluoride membrane (Millipore). After blocking with 5% nonfat milk, the bands were subsequently incubated with primary antibodies specific to Rac1‐Total (1:1000), Rac1‐GTP (1:1000), aggrecan (1:1000), collagen II (1:1000), Sox‐9 (1:1000), ADAMTS5 (1:1000), MMP13 (1:1000), β‐catenin (1:1000), SFRP1 (1:1000), lamin b (1:1000) and β‐actin (1:1000) overnight at 4°C, followed by incubation with the appropriate secondary antibodies. Last, the intensity of the bands was visualized and analysed using Image Lab 3.0 software (Bio‐Rad).The proteins of EPCs were extracted using ice‐cold RIPA with 1 mmol/L phenylmethanesulphonyl fluoride (PMSF), and the protein concentration of samples was measured using the bicinchoninic acid method through BCA protein assay kit (Beyotime). The proteins of EPCs were separated by 8%‐12% .2.7Total RNA of EPCs was extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. cDNA was synthesized using 1 μg RNA through the One Step RT‐PCR Kit (TaKaRa). Quantitative real‐time PCR was analysed using the iQTM SYBR Green Supermix PCR kit through the iCycler apparatus system (Bio‐Rad). All primer sequences were listed as follows: for aggrecan, Forward: 5′‐AAGTGCTATGCTGGCTGGTT‐3′, Reverse: 5′‐GGTCTGGTTGGGGTAGAGGT‐5′; for collagen II, Forward: 5′‐CTCAAGTCGCTGAACAACCA‐3′, Reverse: 5′‐GTCTCCGCTCTTCCACTCTG‐3′; for MMP13, Forward: 5′‐CCAGAACTTCCCAACCAT‐3′, Reverse: 5′‐ACCCTCCATAATGTCATACC‐3′; for ADAMTS5, Forward: 5′‐GGGAATAAGTACTGGGCTGTTCAG‐3′, Reverse: 5′‐CCTCAGAAAGAGCAGCATCGATATG‐3′; for Axin2, Forward: 5′‐ GAACCTGAAGGATCGCAAAA3′, Reverse: 5′‐ GGTTTCAGCTGCTTGGAGAC‐3′; for LEF‐2, Forward: 5′‐ CTGAGCGAGTTGTGTACCG‐3′, Reverse: 5′‐AGTTCCTCCGAAAATGTAGGG‐3′; for β‐actin, Forward: 5′‐TCTCCTCTGACTTCAACAGCGAC‐3′, Reverse: 5′‐CCCTGTTGCTGTAGCCAAATTC‐3′.2.8w/v) pentobarbital (40 mg/kg) and randomly divided into three groups: control group (n = 8 each group), IVDD group and IVDD + NSC23766 group. The coccygeal intervertebral space Co7‐8 was located by palpation and confirmed with an X‐ray radiograph which was selected for the further study. After the rat tail skin was sterilized with iodophor, needles (26G) were applied to perpendicularly puncture the whole layer of annulus fibrosus though the tail skin and needles were maintained for 1min in the disc. After surgery, the rats in IVDD + NSC23766 group were intraperitoneal injected with NSC23766 (50 mg/kg) while other rats were administered with the same amount of saline every 2 days until the rats were sacrificed.Adult Sprague Dawley rats  were anaesthetized with 2%  due to the protocol. Images were visualized and captured under a microscope.2.10v/v) H2O2 for 15 minutes. The sections were incubated with 0.4% pepsin in 5mM HCl for 20 minutes at 37℃ for antigen retrieval, followed with incubation of 5% bovine serum albumin for 30 minutes. After incubation with primary antibody overnight at 4°C, the sections were finally incubated with HRP conjugated secondary antibody. Images were visualized and captured under a microscope. The rate of positive cells during each section was analysed by a separate group of experienced histology researchers who were blinded to the experimental groups.The sections(5 μm) were deparaffinized in xylene and rehydrated in ethanol, and endogenous peroxidase was blocked by 3%  of the coccyx was performed to evaluate the IVDD. The IVDD signal and structural changes in sagittal T2‐weighted images using a 3.0‐T clinical magnet (Philips Intera Achieva 3.0MR) were assessed through MR imaging. The parameters of T2‐weighted in the sagittal plane were as following: fast spin echo sequence with time to repetition (TR) of 5400ms and time to echo (TE) of 920 ms; 320 (h) 9256 (v) matrix; field of view 260; and four excitations. The section thickness was 2 mm with a 0‐mm gap. The degree of IVDD during MRIs images was evaluated by another three orthopaedic researchers in a blinded manner according to the Pfirrmann grade.2.12P < .05 was considered statistically significant.The results are expressed as the mean ± SD Statistical analysis was performed using SPSS statistical software program 20.0. Data were analysed by one‐way analysis of variance (ANOVA) followed by the Tukey's test for comparison between the two groups. 33.1In order to illuminate the relationship between CEP degeneration and Rac1, we compared the expression of Rac1 during normal and degenerated human EPCs and evaluated the expression of Rac1 in IL‐1β‐induced rats EPCs by Western blotting analysis. According to the results, the level of Rac1‐GTP was markedly increased in degenerated EPCs compared with normal chondrocytes in protein level  was taken at 8 weeks to evaluate the degree of disc degeneration in rats. From MRI results, we found higher T2‐weight signal intensity of the intervertebral disc in NSC23766‐treated group than that in IVDD group (Figure Based on the vitro study results, the effect of NSC23766 on the Wnt/β‐catenin signalling in vivo was further verify. Consistent with the vitro findings, immunohistochemical staining and its corresponding quantification demonstrated that NSC23766 could inhibit the β‐catenin expression Figure A,B. Coll4This study was designed to clarify the effect of Rac1 activity in CEP degeneration and disc degeneration and seek for the effective strategy for prevention of IVDD development. The present work, for the first time, illustrated the aberrant activation of Rac1 in CEP degeneration, and Rac1 inhibitor NSC23766 protected the disc from IVDD through CEP regulation via Wnt/β‐catenin pathway.The Rho family of small GTPases has been proved to play critical role in numerous aspects of cell biology, including cell proliferation, apoptosis, migration and invasion.Wnt/β‐catenin signalling has been proved as a crucial factor in regulating several biological processes such as cell fate determination during embryogenesis, cell proliferation, differentiation and apoptosis.The relationship between the Rac1 and Wnt/β‐catenin signalling has been thoroughly studied to date. Wu et al have demonstrated that Rac1 activation could cause the elevated catenin level and nuclear translocation and contribute to aberrant activation of canonical Wnt signalling in cancer cells, indicating the important roles of Rac1 in β‐catenin nuclear localization and β‐catenin‐dependent transcriptional activation.Axin2 and LEF‐1 are one of the major downstream factors of Wnt/β‐catenin signalling. Our data showed that mRNA expression of Axin2 and LEF‐1 was markedly reduced in NSC23766 treatment group than the IL‐1β‐stimulated group. Taken together, these results indicate that the protective role of Rac1 inhibition in rat EPCs may attribute to the regulation of Wnt/β‐catenin pathway.Based on the beneficial effects of NSC23766 in rat EPCs, rat annulus needle puncture models of IVDD were introduced to evaluate the therapeutic value of Rac1 inhibition in in vivo study. Interestingly, in addition to endplate cartilage, the nucleus pulposus was also better preserved in the NSC23766‐treated group than in the IVDD group, indicating that NSC23766 may contribute to the prevention of IVDD also through regulating nucleus pulposus. In addition, our study showed that NSC23766 reduced the β‐catenin expression both in endplate cartilage and nucleus pulposus, suggesting that NSC23766 alleviated the development of IVDD by targeting Wnt/β‐catenin signalling.In conclusion, our research revealed that expression of Rac1 was increased in human‐degenerated EPCs in vivo and IL‐1β‐stimulated rat EPCs in vitro. Rac1 inhibitor NSC23766 could suppress the degradation of EPCs through the regulation of Wnt/β‐catenin signalling. Moreover, our in vivo experiments demonstrated that NSC23766 may ameliorate the development of CEP and disc degeneration in IVDD model. Thus, inhibition of Rac1 could be a effective therapeutic strategy for IVDD treatment.The authors declare no conflict of interests.YSW, CJ and ZMS contributed to conceptualization; DCZ contributed to methodology; QG contributed to software; JJX and JHL contributed to validation; ZXC and ZMS contributed to formal analysis; DCZ and JHL contributed to data curation; CJ and ZMS contributed to writing‐original draft preparation; YSW contributed to writing‐review and editing; ZMS and ZXC contributed to supervision; QG, JJX and ZXC contributed to project administration; YSW contributed to funding acquisition. Click here for additional data file.


Bovine parainfluenza virus type 3 (BPIV3) is one of the important viral respiratory agents associated with the bovine respiratory disease complex (BRDC) in cattle. Previous study has demonstrated that infection of BPIV3 causes innate immune response within the host cell. β-catenin is a key component of the Wnt/β-catenin signal pathway which is involved in the regulation of interferon-beta (IFN-β) transcription. Some viruses can activate while others can inhibit the Wnt/β-catenin signaling pathway. However, the role of β-catenin in BPIV3 infection remains unclear.Here we found that the expression of β-catenin mRNA was up-regulated and β-catenin protein was down-regulated after BPIV3 infection in MDBK cells. Moreover, it was confirmed that overexpression of β-catenin suppressed BPIV3 replication and knockdown of β-catenin promoted viral replication, suggesting that β-catenin inhibits BPIV3 replication. Furthermore, IFN-β signal pathway and virus titer analysis using the GSK3β inhibitor (LiCl) revealed that Wnt/β-catenin can serve as a mechanism to suppress virus replication in infected cells. The results indicated that LiCl promoted the expression and accumulation in the nucleus of β-catenin, which further promoted the expression of IFN-β and OSA1 and suppressed BPIV3 replication. Most importantly, BPIV3 down-regulating β-catenin protein expression was due to degradation of GSK3β mediated proteasome pathway.In summary, we discovered the relationship between β-catenin and BPIV3 replication. These results provided further insight into the study of BPIV3 pathogenesis. Bovine parainfluenza virus type 3 (BPIV3), also known as Bovine respirovirus 3, belongs to the Paramyxoviridae Respirovirus genus. BPIV3 is a single-strand negative-sense RNA virus that enveloped and non-segmented . The BPIProtecting host cells from virus invasion and infection is an important role of antiviral innate immunity , 6. DuriWnt/β-catenin signaling pathway is not only an important way to regulate the early development of embryonic, maintain tissue homeostasis in adults and central nervous system regeneration, but also closely related to cell proliferation, differentiation, migration and apoptosis –14. The Several studies have showed the interaction of the Wnt/β-catenin signaling pathway with viruses, such as the human immunodeficiency virus (HIV), the porcine circovirus-like virus P1, the Human Cytomegalovirus (HCMV), the hepatitis C virus (HCV) and so on. Some viruses can activate while others deactivate the Wnt/β-catenin signaling pathway. For example, HIV suppresses Wnt/β-catenin signaling transduction by interacting its viral negative regulatory factor with β-catenin , HCMV inIn this study, we first investigated the effect of Wnt/β-catenin during BPIV3 infection, and found that the expression of β-catenin mRNA and protein levels was up-regulated and down-regulated respectively during BPIV3 infection. BPIV3 replication was also either suppressed or promoted with overexpression or knockdown of β-catenin gene. Furthermore, under the effect of GSK3β inhibitor LiCl, the expression and accumulation of β-catenin in the nucleus were promoted, and the expressions of IFN-β and OSA1 were further promoted to suppress BPIV3 replication. Further studies revealed that BPIV3 down-regulated β-catenin protein expression due to degradation of GSK3β mediated proteasome pathway. Our findings largely provide some novel insights into virus and host interactions during BPIV3 infection.To explore whether BPIV3 infection affected the expression of β-catenin, Madin-Darby Bovine Kidney Cells (MDBK) were infected with BPIV3 1MOI) at different time points, the level of β-catenin mRNA was determined by qRT-PCR and the expression of β-catenin protein was detected by western blot. The results showed that the level of transcription of β-catenin gene significantly increased at different time points after BPIV3 infection compared to the control group . It was found that the amounts of infectious virus at 24 h decreased at about 8-fold in β-catenin-overexpression cells compared to control group cells  binding sites after entering the nucleus . Given tIn order to verify the effect of β-catenin on IFN-β expression in reverse, post-infection results in β-catenin-knockdown cells were also monitored. As shown in Fig. Given that our results showed significant post-BPIV3-infection decrease in β-catenin’s expression at 36 h Fig. , it is hβ-catenin is a key component in the activation of Wnt/β-catenin pathway and thus plays an important role in antiviral immune response. It can directly or indirectly inhibit the replication of HIV, HCMV, HCV and other types of viruses or control their pathogenicity –26. HoweSpecifically, in the activated Wnt/β-catenin pathway, the degradation complex of β-catenin, AXIN/APC/GSK3β/CK1 complex is depolymerized . β-catenMany other viral infections have been shown to correlate with the functionality of β-catenin. For example, both Porcine Circovirus-like virus and Human Cytomegalovirus inhibit the classical Wnt signal pathway post-infection , 31. WheMultiple studies have shown that different virus may employ different mechanisms to manipulate the process of Wnt/β-catenin pathway’s participation in viral replication , 21, 31.In summary, results of our study indicate that β-catenin plays a significant role in BPIV3 infection. BPIV3 infection suppresses the protein level of β-catenin, since β-catenin can affect viral replication by promoting the expression of IFN-β and ISGs. Moreover, viral invasion mostly down-regulates the amount of β-catenin by hijacking its GSK3β mediated proteasome degradation. Overall, our study showed the important effect of β-catenin on BPIV3 infection and prompts us to do more in the future to reveal its comprehensive involvement in virus-host interaction.50 of BPIV3 was determined by the Reed-Muench method, which was described previously [BPIV3 strain was isolated and stored in the Ruminant Disease Research Center, Shandong Normal University, Jinan, Shandong Province, China. TCIDeviously , 36.2 atmosphere.The 293 T (GDC0067) originating from China Center for Type Culture Collection (CCTCC) and MDBK cell were provided by American Type Culture Collection (ATCC No.: CCL-22). MDBK cells and 293 T cells were preserved in the Ruminant Disease Research Center, Shandong Normal University, and cultured in Dulbecco’s Modified Eagle’s Medium  and supplemented with 10% fetal bovine serum  at 37 °C in a humidified, 5% COAbcam. The secondary antibodies used for western blot were HRP-linked mouse IgG  from Santa Cruz Biotechnology.Anti-β-catenin antibody from Santa Cruz Biotechnology  was used for immunofluorescence and western blot. Anti-β-actin, anti-Lamin B1, anti-GSK3, and anti-Flag antibodies from Santa Cruz Biotechnology  were used for western blot. The secondary antibodies used for immunofluorescence were FITC-conjugated goat anti-mouse IgG  from -△△Ct), and β-actin mRNA expression levels were used for normalization process [MDBK cells were infected with BPIV3 at a MOI of 1, and total cells were collected at 12 h, 24 h and 36 h post infection to detect the differential expression of β-catenin using specific primers as shown in Table  process .Table 1Xba I) and β-catenin-R (BamH I) shown in Table https://rnaidesigner.thermofisher.com/ rnaiexpress/design.do), synthesized by TSINGKE Biological Technology Co., Ltd.  and cloned into the pYr-Lvsh lentiviral vector and named β-catenin-shRNA1/2/3, respectively [The PCR primer β-catenin-F  and then subjected to western blot analysis. β-actin and Lamin B1 were used as internal references of cytoplasmic and nucleus protein, respectively.MDBK cells infected by BPIV3 or pretreated by LiCl or not were subjected to 10% SDS gel electrophoresis and transferred onto PVDF membranes . The membranes were then blocked using 8% non-fat dry milk dissolving in TBST for 2 h, incubated with primary antibodies at a dilution of 1:2000 for 2 h at room temperature. Afterwards the membranes were washed by TBST and incubated with secondary antibodies for 1 h at room temperature. Finally, P-ECL Chemiluminescence Kit  was used to show the target protein band using chemiluminescence imaging system .MDBK cells in good condition were dissolved to form a cell suspension and seeded in 24-well plates containing the coverslips. When cells reached 50% confluence in the coverslips, LiCl at 20 mM was added for 24 h. Next, the original culture was discarded, MDBK cells were washed with phosphate-buffered saline (PBS), fixed with 4% paraformaldehyde /PBS for 15 min at room temperature, washed with PBS and permeabilized with 1% Triton X-100 /PBS for 10 min. Subsequently, cells were blocked with 0.5% bovine serum albumin , incubated with the corresponding primary antibodies diluted in BSA for 1 h at room temperature. Cells were then washed with PBS and incubated with the corresponding secondary antibodies for 1 h at room temperature. After the 4′,6-diamidino-2-phenylindole  was added to dye nucleus for 5 min without light, cells were observed with a confocal microscope and recorded.P value below 0.05 was considered statistically significant .Graphpad prism software  was used to perform statistical analysis. Results were presented as the mean values ± standard deviation (SD) from at least three independent experiments, and


This paper reported the effect of high temperature on the electro-mechanical behavior of carbon nanotube (CNT) reinforced epoxy composites. CNT/epoxy composites were fabricated by dispersing CNTs in the epoxy matrix using a solution casting method. Electrical conductivity measurements obtained for the CNT/epoxy composites indicated a steadily increasing directly proportional relationship with CNT concentration with a percolation threshold at 0.25 wt %, reaching a maximum of up to 0.01 S/m at 2.00 wt % CNTs. The electro-mechanical behavior of CNT/epoxy composites were investigated at a room temperature under the static and cyclic compressive loadings, resulting that the change in resistance of CNT/epoxy composites was reduced as increasing CNT concentration with good repeatability. This is due to well-networked CNTs conducting pathways created within the solid epoxy matrix observed by scanning electron microscopy. Temperature significantly affects the electro-mechanical behavior of CNT/epoxy composites. In particular, the electro-mechanical behavior of CNT/epoxy composites below the glass transition temperature showed the similar trend with those at room temperature, whereas the electro-mechanical behavior of CNT/epoxy composites above the glass transition temperature showed an opposite change in resistance with poor repeatability due to unstable CNT network in epoxy matrix. In recent years, carbon nanotubes (CNTs) have attracted considerable interest for many industrial applications ,2,3. CNTThe strain sensing capability of CNT nanocomposites is considered to be a key requirement for future structural health monitoring techniques and applications. To overcome these problems and requirements, some studies have tried to develop novel self-sensing nanocomposites for these types of applications ,15,16,17The effect of temperature on CNT reinforced composites has been studied for different filler-resin composites. Gojny and Schulte  investigA filler material used for the CNT/epoxy composite was high purity (>95.0 wt %) and chemical vapor deposition grown multi-walled carbon nanotubes from US Research Nanomaterials Inc. . The average diameter and length of CNTs were 4–10 nm and 50 µm, respectively. A low viscosity epoxy resin type (IN2 Epoxy Infusion Resin) combined with a hardener (AT30 slow) were obtained from Easy Composite . The viscosity of the matrix resin was 200–450 mPas and a pot life was 80–100 min at a room temperature. The solvent used for dispersion of CNTs was high purity of acetone (> 95%) supplied by Acros Organics Ltd . The CNT/epoxy composites were fabricated by uniformly dispersing the CNTs in the epoxy matrix as described in authors’ previous studies ,22,23. Fσ) of samples was calculated using Equation (1).R is the electrical resistance of the sample, A and L are a cross-sectional area and the length of the samples, respectively. The compression testing of the specimens was carried out using a universal testing machine . In order to investigate the effect of temperature on the electromechanical behavior of the specimens, the static and cyclic compressive loading was applied to the specimens at a displacement control of 0.5 mm/min. Temperature was controlled by placing the specimens in a heater box, as shown in A fracture surface of the specimen was observed using a scanning electron microscope  at 30 kV to assess the dispersion of CNTs in the matrix. The samples were coated with a thin gold film using sputter coating  for 10 min. For the electrical conductivity, two different multimeters  was used to record resistance. To minimize measurement errors due to the contact resistance between the tip of test probe, both electrodes of the specimen was painted with high purity silver, eliminating an issue for the contact resistance to use two-probe method. Two-probe method was applied for the measurement of the electrical resistance for our specimens because of simple experiment. The electrical conductivity  created within the matrix material as shown in −2 S/m at 2.5 wt % CNTs. The experimental data were also analyzed using a percolation theory, The electro-mechanical response of CNT/epoxy composites was investigated under both static and cyclic compressive loadings. Temperature generally affects the materials properties of CNT reinforced composite ,26,27. Fg) of 65 °C, Tg significantly affects the electro-mechanical response of CNT/epoxy composites. It can be speculated, shown in the g would increase in the inter-distance between CNTs and thus regenerate CNT network, destroying conductive pathways and thus increasing resistance under the compressive loadings.Although there has been much research on the effect of temperature on electrical properties of carbon nanotube reinforced composites at rest, there has been no research on the electro-mechanical behavior of those materials under the different temperatures. Therefore, we studied the electro-mechanical behavior of CNT/epoxy composite with 65 °C of the glass transition temperature  under stThe present study investigated the electro-mechanical behavior of carbon nanotube reinforced epoxy composites at various temperature. It was found that temperature significantly affects the electro-mechanical behavior of CNT/epoxy composites. In particular, the CNT/epoxy composites exposed to above glass transition temperature showed an opposite and unrepeatable electro-mechanical behavior under the compressive loadings compared to those exposed to below glass transition temperature. CNT/epoxy composites may enable potential applications to sensors and sensor-integrated materials. However, the influence of temperature on the electro-mechanical behavior for CNT/epoxy composite must be considered.


Visualizing actin filaments in fixed cells is of great interest for a variety of topics in cell biology such as cell division, cell movement, and cell signaling. We investigated the possibility of replacing phalloidin, the standard reagent for super-resolution imaging of F-actin in fixed cells, with the actin binding peptide ‘lifeact’. We compared the labels for use in single molecule based super-resolution microscopy, where AlexaFluor 647 labeled phalloidin was used in a dSTORM modality and Atto 655 labeled lifeact was used in a single molecule imaging, reversible binding modality. We found that imaging with lifeact had a comparable resolution in reconstructed images and provided several advantages over phalloidin including lower costs, the ability to image multiple regions of interest on a coverslip without degradation, simplified sequential super-resolution imaging, and more continuous labeling of thin filaments. Super-resolution methods are optical microscopy methods that surpass the diffraction limit of light, either by manipulating the illumination pattern like STED  and SSIMA common target in super-resolution is the actin cytoskeleton. Actin exists in cells as either free, monomeric globular actin (G-actin) or filamentous actin (F-actin) , 11. TheAmanita phalloides that binds with high specificity to F-actin and prevents filament depolymerization [−4 1/s [Phalloidin is a heptapeptide derived from the poisonous mushroom rization , 26. Dir [−4 1/s , leading [−4 1/s , 28.Lifeact is a short peptide taken from the first 17 amino acids of the yeast Abp140 and can be expressed in cells linked to a fluorescent protein to visualize F-actin dynamics . LifeactIn this article, we compare the quality of lifeact in single molecule super-resolution imaging to phalloidin labeling. To image phalloidin, we used the dSTORM technique  where ph°C and 5% carbon dioxide. Rat Basophilic Leukemia (RBL-2H3) cells were cultured in MEM supplemented with 10% heat-inactivated fetal bovine serum, 5 U/ml penicillin, 0.05 mg/ml streptomycin, and 2mM L-glutamine at 37 °C and 5% carbon dioxide. For imaging, cells were plated overnight on 25 mm coverslips .HeLa and RBL-2H3 cells were originally purchased from ATCC. HeLa cells were authenticated by short-tandem repeat analysis. RBL-2H3 cells are routinely checked for binding of IgE and the ability of the cells to degranulate. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium  supplemented with 10% cosmic calf serum (HyClone), 5U/ml penicillin, 0.05 mg/ml streptomycin, and 2 mM L-glutamine  and maintained at 37 2 (PEM) with pH 7.2. Briefly, cells were washed with warm PEM and fixed in 0.6% paraformaldehyde with 0.1% glutaraldehyde and 0.25% Triton diluted in PEM buffer for 60 seconds and followed by a hard fixative buffer including 4% paraformaldehyde and 0.2% glutaraldehyde diluted in PEM for two hours. Cells were washed 2x in PBS, incubated for 10 min in 0.1% NaBH4 for 10 min to reduce background fluorescence due to glutaraldehyde, and followed by a 2x wash with PBS. To quench reactive cross-linkers, the samples were incubated in 10 mM Tris for 10 min, followed by 2 washes with PBS. Finally, samples were permeabilized by incubation for 15 min in 5% BSA and 0.05% Triton X-100 diluted in PBS. At the end, samples were washed 1x with PBS and prepared for the labeling process.Samples were fixed using a cytoskeleton-preserving buffer composed of 80 mM Pipes, 5 mM EGTA, and 2 mM MgClTo label actin using phalloidin, fixed cells were incubated for an hour in 0.56 μM AF647-conjugated to phalloidin  in PBS. The samples were washed once in PBS and placed in dSTORM imaging buffer. The dSTORM buffer included an enzymatic oxygen scavenging system and primary thiol: 50 mM tris, 10 mM NaCl, 10% w/v glucose, 168.8 U/ml glucose oxidase , 1404 U/ml catalase , and 60 mM 2-aminoethanethiol (MEA)  with pH 8.0, to provide a suitable chemical condition for having a photo-switchable AF647 dye.For lifeact labeling, we used a customized 17-amino-acid peptide conjugated to Atto 655, ). For imaging, lifeact was diluted to 0.7 nM in an optimized imaging buffer including 10 mM HEPES, 150 mM NaCl, 10% glucose, and 0.1% BSA with pH 7.0 for labeling the actin filaments.25 mm coverslips were mounted in an Attofluor cell chamber . The corresponding imaging buffer for each approach was added and a clean 25 mm coverslip was used to seal the chamber to avoid oxygen permeation into the buffer.et al. [2) for 5 min to photobleach the sample, followed by a 20 min incubation in 0.1% NaBH4 to quench any remaining active AF647 and finally washed 2x with PBS. To remove lifeact-Atto655, the sample was washed 12 x 1 min with 1 mM HEPES, 150 mM NaCl, 5% glucose, and 0.1% BSA in diH2O. In both cases, the sample was re-labeled with anti-α-tubulin-AF647 at 2.5 μg/ml for an hour in 2% BSA, 0.05% Triton diluted in PBS, followed by 3x wash with 2% BSA and 0.05% Triton in PBS for 5 min each. For data collection, the samples were kept in dSTORM buffer and sealed with a 25 mm coverslip. Re-alignment of the sample was done using a brightfield reference image as described by Valley et al. [Sequential super-resolution was performed in a manner similar to that described by Valley et al. . First, y et al. .x-y manual stage was installed on the microscope for cell location and brightfield registration. A mounted LED with a wavelength of 850 nm  was used for brightfield illumination. Brightfield images were collected on a CMOS camera  after reflection by a short-pass dichroic beam splitter  and then passing through a single-band bandpass filter . A 638 nm laser  was coupled into a single-mode fiber and reflected by a dichroic beam splitter  then focused onto the back focal plane of the 1.49 NA objective lens . Emission for super-resolution data was collected through a short-pass dichroic beam splitter  and a single-band bandpass filter  on an EMCCD camera . All the instruments were controlled by custom-written software in MATLAB (MathWorks Inc.) [2 in TIRF  illumination with an approximate exposure time of 40 ms for lifeact and 10 ms for phalloidin (see Exposure time optimization). Brightfield registration was employed to correct for drift after every 3000 frames for phalloidin experiments and 2000 frames for lifeact experiments, as described in [The samples were mounted on the stage of a microscope with a custom-designed chamber holder. The imaging system was built on an inverted microscope . A nano positioning stage  mounted on an ks Inc.) . Imagingribed in .All analyses were performed using a custom-written single molecule analysis (SMA) software package in MATLAB combined with the Statistics Toolbox  and DIPimage . For 2D Q) assuming Poisson statistics. All analyses were accomplished using the MATLAB software developed by Nieuwenhuizen et al. [http://www.diplib.org/add-ons.To estimate the resolution from the super-resolution images of phalloidin and lifeact, we used Fourier Ring Correlation (FRC) , which mn et al.  found atoff, where koff is the rate of emitters going from on to off in units of 1/ms  containing a segment of filament were selected manually. The selected segments were rotated to be along the We employed the two actin labeling approaches using phalloidin and lifeact and compared the super-resolution images of actin structures in fixed HeLa and RBL-2H3 cells. The reversible binding of lifeact generates a small percentage of spurious localizations from transient, non-specific binding. These appear as isolated localizations away from any filaments. Localizations that did not have at least four other localizations within 15 nm were removed before further analysis see , S1 Fig.We estimated the thickness and continuity of actin filaments found in images from each condition. As illustrated in It is often desired to collect data from multiple cells or regions on one coverslip. To investigate this aspect, we imaged actin structures for an extended period using both labeling methods. We found that the lifeact approach provided a near constant number of localizations per frame, whereas the number of localizations per frame with phalloidin imaging dropped considerably even during the imaging of the first cell on a coverslip . This caImaging of multiple structures within the same cell is of interest in order to reveal a spatial correlation between the cellular structures. Here, we compared phalloidin and lifeact strategies in their capacity for sequential super-resolution imaging . As depiIn evaluating super-resolution imaging results of the lifeact and phalloidin labeling methods, we considered four aspects: image resolution, filament thickness and continuity, multiple dataset collection, and capability for sequential imaging. The resolution as measured by FRC analysis showed that the average resolution in the case of lifeact is comparable or to that with phalloidin, with phalloidin having a small advantage in the RBL cells but not in the HeLa cells. It is important to note that the FRC resolution measure gives an average resolution over the image, but cannot directly assess the quality of labeling.Targeting the actin binding sites depends on the biochemical structure of each actin-detecting probe and the various actin architectures . For exaThe blinking mechanism in the lifeact method depends on the binding/unbinding of the peptide. This process benefits from a bright, photostable dye, such as Atto655 so that it doesn’t photobleach as it diffuses into the TIRF field, binds actin, is imaged for ~ 0.1 s, and then unbinds a diffuses away. The large pool of dye-labeled lifeact provides for consistent localizations over long imaging periods as the probes are replenished from the buffer pool. However, the background fluorescence from the fluorescent, diffusing lifeact requires a TIRF or near-TIRF modality to reduce background, restricting imaging to near the coverslip. Phalloidin is semi-permanently bound to actin with a dissociation rate of a few hours. In this case, the blinking mechanism is provided by light-activated photoswitching of the AF647 label. Because the label is not replenished, it invebitaly either undergoes photobleaching during data collection  or is loBoth methods were successful with the use of sequential labeling to image actin and microtubules within the same cell. Lifeact had the advantage that the probe could simply be washed out before microtubule labeling. After imaging phalloidin, the additional steps of photobleaching and quenching were required to eliminate crosstalk with the first imaging cycle.In summary, we found that lifeact provides a reliable and inexpensive alternative to phalloidin for actin visualization in fixed cells, particularly for super-resolution imaging near the coverslip. Imaging with lifeact additionally confers several advantages over phalloidin including more continuous labeling of filaments, the ability to image multiple cells per sample and simplification of sequential super-resolution imaging.S1 Fig(A) Reconstructed super-resolution images of actin filaments in HeLa cells labeled with lifeact-Atto655 (top) and phalloidin-AF647 (bottom). (B) Super-resolution images of actin filaments in HeLa cells labeled with lifeact-Atto655 (top) and phalloidin-AF647 (bottom) after removing localizations that had less than 4 other localizations within 15 nm. Scale bars are 500 nm.(TIF)Click here for additional data file.S2 FigData was collected with six exposure times: 25 ms, 30 ms, 33 ms, 35 ms, 38 ms, and 45 ms. The distribution of the number of connected localizations per binding event (blue) fits with an exponential model (red dashed line) to extract a binding lifetime.(TIF)Click here for additional data file.S3 Fig(A) Super-resolution images from two lifeact tagged samples. (B) Super-resolution image from two phalloidin tagged samples. The scale bars in full field images are 500 nm.(TIF)Click here for additional data file.S4 FigSingle filaments were cropped from the reconstructed super-resolution images from lifeact-Atto655 (top) and phalloidin-AF647 (bottom). The filaments were reconstructed using localizations from the first (A) 30,000, (B) 60,000 and (C) 100,000 frames. The scale bars are 20 nm. (D) The cumulative number of localizations per frame from data collected using lifeact (red) and phalloidin (blue). Data is from HeLa cells.(TIF)Click here for additional data file.S5 FigPlots show the number of localized single molecules per frame for complete data collections for individual cells. (A) HeLa cells labeled with lifeact-Atto655. (B) RBL-2H3 cells labeled with lifeact-Atto655. (C) HeLa cells labeled with phalloidin-AF647. (D) RBL-2H3 cells labeled with phalloidin-AF647.(TIF)Click here for additional data file.