The structural and morphological properties of the synthesized composite materials were characterized via Raman spectroscopy, XRD, SEM, and SEM–EDX, and the morphological properties of the composite materials were confirmed by the nanostructured composite at the nanometer scale. The CuO@MnO2 and CuO@MnO2/N-MWCNT composite electrodes were fabricated in a three-electrode configuration, and electrochemical analysis was performed via CV, GCD, and EIS. The composite electrodes exhibited the specific capacitance of ~ 184 F g−1 at 0.5 A g−1 in the presence of a 5 M KOH electrolyte for the three-electrode supercapacitor application. Furthermore, it exhibited significantly improved specific capacitances and excellent cycling stability up to 5000 GCD cycles, with a 98.5% capacity retention.Nitrogen-doped multiwalled carbon nanotubes (N-MWCNTs) have been used to fabricate nanostructured materials for various energy devices, such as supercapacitors, sensors, batteries, and electrocatalysts. Nitrogen-doped carbon-based electrodes have been widely used to improve supercapacitor applications via various chemical approaches. Based on previous studies, CuO@MnO In particular, supercapacitors have received considerable attention owing to their excellent life cycles and high-power density results. Supercapacitors can be categorized as electric double-layer capacitors (EDLC) and pseudocapacitors, depending on their complex electrochemical reaction. In the case of the EDLCs, energy is collected by the electrostatic adsorption of charges on the electrode surface in the parallel plate capacitor. In pseudocapacitors, energy is collected via reversible Faradaic responses on the material electrode surface8.Recently, electronic devices for storage applications comprising nanometer-scale materials with excellent capacitance and cyclic stability are being rapidly developed. Numerous methods for fabricating electrode materials for practical applications have been reported in various supercapacitor, battery, and fuel-cell studies11. An alternative approach is to use potential electrode materials such as Co3O4, MnO2, NiO, CuO, Fe3O4 and V2O5, which have been widely used in supercapacitor applications15. The higher specific capacitance of these materials is because of the presence of metal oxides, which exhibit more pronounced redox behavior than the carbon in carbon-based electrodes; thus, a specific capacitance and excellent electrochemical stability are realized via irreversible reactions18. Notably, copper is an excellent electrode material because it is nontoxic, abundantly available, low-cost, and easy to fabricate into an electrode for supercapacitor applications19. Zhang et al. reported that flower-like CuO nanostructured materials yielded a capacitance value of 134 F g−1 in the presence of a 1 M KOH electrolyte18. In addition, mesoporous copper oxide nanoribbons were fabricated on nickel foam electrodes, with a capacitance of 137 F g−1. Wang et al. fabricated nanosheets with a capacitance of ~ 569 F g−119.Carbon-based electrode materials are an appropriate choice for supercapacitor applications because of their excellent specific capacitance and power density2 has emerged as a promising electrode material for supercapacitor applications owing to its high theoretical specific capacitance, high electrochemical activity, and environmental friendliness22. However, the low conductivity (10−5 to 10−6 S cm−1) and unstable structure of MnO2, which causes poor electrochemical cyclability, limits its application. To address these limitations and improve the electrochemical behavior of supercapacitors, several studies have attempted to design nano-MnO2 structures. It has been reported that the electrochemical performance of MnO2 depends on its morphology, surface area, and crystal structure. Zhang et al. reported MnO2-based electrodes for supercapacitors with different crystal structures and morphological behaviors synthesized via a hydrothermal process. Nano-a-MnO2 ball with a low degree of crystallinity exhibited a high specific capacitance of 200 F g−1 and excellent cyclic stability. Rusi et al. also synthesized a-MnO2 with a low-crystalline electrode via electrochemical deposition using a manganese acetate tetrahydrate electrolyte. The assembled MnO2 electrode, characterized via a cyclic voltammetry test in the Na2SO4 electrolyte at a scan rate of 1 mV s−1, exhibited a high specific capacitance of 238 F g−1 and excellent stability, with an 84% capacitance retention over 1900 cycles. By contrast, CuO, which is an active material that contributes to pseudocapacitance, possesses a high specific surface area, is highly conductive and environmentally friendly, and can be incorporated with MnO2. Furthermore, the synergetic effect also plays an important role in improving the performance of the MnO2–CuO composite on the carbon surface. Therefore, a material comprising both MnO2 and CuO will exhibit better electrochemical performance, conductivity, cycling stability, and morphological properties than individual MnO2 and CuO materials. Therefore, this study focused on CuO@MnO2 and CuO@MnO2/N-multiwalled carbon nanotube (MWCNT) composites synthesized via a hydrothermal reaction process using a three-electrode configuration for supercapacitor applications. The nanocrystalline structure and morphological properties of the resulting composites were investigated via FE-SEM. In addition, the electrochemical properties of the composite electrodes were analyzed using CV, GCD, and EIS in the presence of a 5M KOH electrolyte.MnO3)2·5H2O, potassium permanganate (KMnO4), manganese (II) acetate (C4H6MnO4), hydrochloric acid (HCl), sulfuric acid (H2SO4), phosphoric acid (H3PO4), ammonia , absolute ethanol (C2H5OH), N-methyl pyrrolidinone (NMP), and polyvinylidene fluoride (PVDF) were obtained from Sigma-Aldrich Chemicals, and the overall electrochemical experiments were performed using double distilled (DD) water.MWCNTs, copper nitrate penta hydrate Cu using a uniform paste. Subsequently, this composite paste was coated uniformly on a strip of a nickel wire (1 × 1 cm−2) current collector and vacuum-dried in an oven at 90 °C for 10 h. The fabricated working electrode, Pt electrode (counter electrode), and a reference electrode (Ag/AgCl) were used to study the electrochemical properties of the synthesized composite materials.The composite was fabricated via a three-electrode configuration and its electrochemical properties were determined using CV, GCD, and EIS analyses. The material (85:15:5) was mixed with 2 and CuO@MnO2/NMWCNT composite materials were characterized using Raman, XRD, SEM, SEM–EDS, TEM, and CV analyses for supercapacitor application. XRD results of the composite materials were obtained using a Rigaku Rotaflex (RU-200B) X-ray diffractometer. The composite materials were analyzed using a He–Ne laser beam in the RM 200 Raman spectral microscope, and the morphological properties of the composite samples were determined using FE-SEM (JEOL) and SEM–EDX analyses. The electrochemical properties of the composite materials were determined based on CV, GCD and EIS results via CHI 7081C .The synthesized CuO@MnO2/N-MWCNT composite material for supercapacitor applications. The peaks marked in green at 28.50°, 37.30°, 42.80°, 56.80°, 59.40°, and 72.50° corresponding to the (100), (101), (111), (113), (200), (220), (202), (400), (211), (220), (330), (400), and (420) planes of MnO2, respectively, matched with the data in PDF file no.44-0141. The 2θ peaks marked in red at 30.50°, 32.50°, 35.50°, 38.70°, 48.80°, 53.25°, 58.65°, 61.50°, 66.20°, 68.10°, 72.40°, and 75.20° corresponded to (110) (111) (202) (112), (020), (202), (113), (311), and (004) planes, respectively. The obtained results agreed with those of the CuO (JCPDS file no. 72-0629) monoclinic structure in the composite materials. In this XRD pattern, CuO@MnO2 was decorated on the N-MWCNT surface. Therefore, the MnO2/CuO in the composite materials was recognized as MnO2 with cubic and monoclinic CuO structure21.Figure −1 were ascribed to the three distinct peaks of the N-MWCNT composite. The D band was assigned to the lattice defect of the phonon mode of vibration from the N-MWCNT surface. The G band signifies the C–C  and double-degenerate phonon modes of Eg2 symmetry. The G band is associated with the vibrational modes of the sp2-bonded carbon atoms in the graphitic layer from the N-MWCNT, whereas the D band is associated with the breathing mode of sp2 bonding, which is present in disordered graphite. We observed that N doping enhanced the D-band intensity of the composite sample. The peak intensity ratio between the D and G bands (ID/IG) was 1.05 for the pristine N-MWCNT [ref], and 1.28 and 1.30, respectively, for the composite samples. This D enhancement indicates that N doping produces lattice defects in the graphitic layers23. The lower wave region of the metal oxides was formed with less intense Ag, Bg1, and Bg2 vibration modes, as discussed previously for metal oxide materials27.The Raman spectra of the composites are shown in Fig. 2 exhibited well-decorated nanotubes with an outer diameter of about ~ 20–30 nm and an inner thickness of ~ 10–20 nm  mV/s results indicate that a similar trend of the electrochemical behaviors and rate capabilities occurred in the presence of the 5 M KOH electrolyte. The obtained results became more pronounced with scan rates and peak shifts toward positive and negative potentials owing to the polarization effect and electron transfer rates between the Cu and Mn oxides on the carbon surface37. The improved electrochemical behavior was strongly related to the chemical composition and morphology of the synthesized composite or 5 M KOH electrolyte for supercapacitor applications39.The electrochemical properties of MnO−1 in the 0.0–0.6 V range, and the results are shown in Figs. 41. The composite electrodes exhibit triangular forms, which indicate reversible redox reactions in the electrochemical process. The outcome capacitance values of CuO@MnO2 were calculated as 61.54, 53.8, 46.15, 27.69 and 26.90 F g−1 at current densities of 0.5, 1.0, 1.5, 2, and 2.5 A g−1, respectively. The CuO@MnO2/N-MWCNT composite yields values of 184, 165,154, 125, and 116 F g−1 at the same current density. The variation of specific capacitance with current density is shown in Figs. −1 for 5000 cycles. The specific capacitance of the composite electrode was 98.5% of its initial value, indicating the excellent stability retention and the performance of the composite electrode materials in terms of enhanced electrochemical properties with a 5 M KOH electrolyte. The unique morphologies of the synthesized composite materials may prevent aggregation and volume expansion during long-term cycling, which is useful for achieving structural stability of the electrodes43.GCD analysis was performed at current densities of 0.5, 1.0, 1.5, 2 and 2.5 A g2 and CuO@MnO2/N-MWCNT composite electrodes resulted from the surface properties and improved morphological properties of the electrodes. The specific capacitances of the CuO@MnO2 and CuO@MnO2/N-MWCNT composite electrode materials were better than those of other electrodes with previously reported materials, as shown in Table 143. The improvement in the electrochemical properties depends on the Faradaic redox reactions of the Cu (I)/Cu(II) and Mn (Mn2+) transitions and increases the capacitances, facilitating supercapacitor applications44. Furthermore, the electrochemical behaviors of the synthesized CuO@MnO2 andCuO@MnO2/N-MWCNT composite electrodes were studied via impedance spectroscopy (EIS) analysis, and the fabricated electrodes of composite (EIS) results are presented in Figs. 47. Furthermore, the Cu2+/Cu+ and Mn2+ to Mn+ redox reactions improve the electrochemical behavior of the electrode materials in the lower wave frequency region, making these composites potentially suitable for supercapacitor applications53.The results revealed that the synthesized composites exhibited superior specific capacitance values compared with those fabricated in previous studies Table . The imp2 and CuO@MnO2/N-MWCNT composites synthesized via a sonication-assisted process and hydrothermal reaction process exhibited excellent morphological properties and improved electrochemical behavior. The moreover, the CuO@MnO2/N-MWCNT composite electrodes exhibited excellent electrochemical properties with a high specific capacitance of 184 F g−1 at a current density of 0.5 A g−1 in the presence of 5 M KOH electrolytes. In particular, a capacity retention of 98.5% at 0.5 A g−1 was maintained over 5000 continuous GCD cycles. This enhancement in the electrochemical properties of the material was attributed to the surface properties and improved morphological behavior of the material. The synthesized composite electrodes exhibited excellent electrochemical, morphological, and cyclic stabilities, rendering them suitable for supercapacitor applications.CuO@MnOSupplementary Figure S1.


The resulting NPs with eight different surfactant–polymer matrix combinations were evaluated for physical properties, oxygen sensitivity, effect of changes in dispersion matrix, and cytotoxicity. The EC NPs exhibited a narrower size distribution and 40% higher sensitivity than PS, with Stern–Volmer constants (svK) 0.041–0.052 µM−1 for EC, compared to 0.029–0.034 µM−1 for PS. Notably, ethyl cellulose NPs protected with PF68 were selected as the preferred formulation, as they were not cytotoxic towards 3T3 fibroblasts and exhibited a wide phosphorescence lifetime response of >211.1 µs over 258–0 µM and ~100 µs over 2.58–0 µM oxygen, with a limit of detection (LoD) of oxygen in aqueous phase of 0.0016 µM. The EC-PF68 NPs were then efficiently encapsulated in alginate microparticles along with glucose oxidase (GOx) and catalase (CAT) to form phosphorescent nanoparticles-in-microparticle (NIMs) glucose sensing microdomains. The fabricated glucose sensors showed a sensitivity of 0.40 µs dL mg−1 with a dynamic phosphorescence lifetime range of 46.6–197.1 µs over 0–150 mg dL−1 glucose, with a glucose LoD of 18.3 mg dL−1 and maximum distinguishable concentration of 111.1 mg dL−1. Similarly, lactate sensors were prepared with NIMs microdomains containing lactate oxidase (LOx) and found to have a detection range of 0–14 mg dL−1 with LoD of 1.8 mg dL−1 and maximum concentration of 13.7 mg dL−1 with lactate sensitivity of 10.7 µs dL mg−1. Owing to its versatility, the proposed NIMs-based design can be extended to a wide range of metabolites and different oxygen-sensing dyes with different excitation wavelengths based on specific application.A highly sensitive, biocompatible, and scalable phosphorescent oxygen sensor formulation is designed and evaluated for use in continuous metabolite sensors for biological systems. Ethyl cellulose (EC) and polystyrene (PS) nanoparticles (NPs) stabilized with Pluronic F68 (PF 68), Polydimethylsiloxane- Precise and reliable methods for the measurement of dissolved oxygen are important in the design of biosensors tailored for different life science applications, including in vivo metabolite monitoring, anaerobe culture, and bioprocess monitoring ,2,3. BeyA key to achieving accurate and bias-free quantitative monitoring of oxygen and metabolites is to design highly sensitive probes that may be deployed in non-invasive or minimally invasive fashion. Needle-based electrochemical and optical sensors have been developed for in vivo applications ,11; whilPhosphorescent oxygen probes carry a unique advantage over electrochemical oxygen sensors as they do not consume oxygen . SensingIn this study, we present a design strategy to realize stable, biocompatible phosphorescent oxygen nanoprobes with high sensitivity and wide dynamic range by encapsulating palladium(II)meso tetra(4-carboxyphenyl)tetrabenzo-porphyrin) (PdBP) in polymeric nanoparticles. We compared properties such as size distribution, sensitivity to oxygen, and toxicity towards 3T3 fibroblasts for different polymeric carriers and surfactants used in synthesis. Further, we examined embedding the nanoprobes along with model enzymes into hydrogel matrices to form nanoparticle-in-microparticle (NIMs) systems and studied their response to metabolite challenges in vitro under simulated physiological conditions to assess potential for continuous monitoring using phosphorescence lifetime readout. These studies identified key characteristics relating chemical and physical properties to function of oxygen-sensitive material and revealed important design considerations for preparation of materials intended for contact with living systems.−1 glucose, and sodium pyruvate) was procured from Corning/Mediatech, Inc., Manassas, VA, USA. Heat-inactivated, fetal bovine serum (FBS) was obtained from Gibco, Grand Island, NY, USA while 100X EmbryoMax® penicillin/streptomycin (P/S) antibiotics and cell counting kit  for cytotoxicity assay were purchased from Millipore Sigma, St. Louis, MO, USA. Lactate oxidase  was purchased from AG Scientific, San Diego, CA, USA. Cyanine5.5 N-hydroxysuccinamide ester  was purchased from Broadpharm, San Diego, CA, USA.Alginate , ethyl cellulose , polystyrene , calcium carbonate (Cat. No. 239216), TRIS base (Cat No. T1503), [2-(Methacryloyloxy)ethyl]trimethylammonium chloride , catalase , calcium chloride (Cat No. 22350), pluronic F 68 , cetyltrimethylammonium bromide , sodium dodecylsulfate , and tetrahydrofuran  were purchased from Sigma-Aldrich, Inc., St. Louis, MO, USA. Trizma hydrochloride  and bis-acrylamide  were purchased from VWR Chemicals LLC, NY, USA. 2-(methacryloxy)ethylphosphorylcholine , glucose oxidase from Aspergillus niger , -sorbitan tri-9-octadecenoate , polyoxyethylenesorbitan trioleate  were purchased from Tokyo Chemical Company Co., LTD, Tokyo, Japan. 2-hydroxyethylmethacrylate  was purchased from Polysciences Inc. Warrington, PA, USA. Palladium (II) meso-tetra(4-carboxyphenyl)tetrabenzo-porphyrin)  was purchased from Frontier Specialty Chemicals, Logan, UT, USA. Iso-octane (Cat No. 94701) was purchased from Avantor performance materials, LLC, Randor, PA, USA. All of the above chemicals were used as obtained without any further purifications. 1X Dulbecco’s Modification of Eagle’s Medium . A JEOL 1200 EX TEM equipped with tungsten filament source at 100 kV acceleration voltage was used for TEM studies. Nanoparticle size distribution in suspensions was determined using the NanoSight LM10 HS nanoparticle tracking analysis (NTA) instrument. Confocal fluorescence microscopy was performed on Olympus FV1000 confocal microscope using 60×/1.2 water immersion objectives. An S25N18G dispersing tool, IKA T25 easy clean digital homogenizer was used to prepare alginate microspheres. A Nexcelom Cellometer Mini device was used for determination of size distribution of alginate microparticles.For oxygen sensitivity (Stern–Volmer) studies, the hydrogel sample punches were held in place on an optically transparent acrylic sample holder using rubber cantilevers. The acrylic holder with samples was then sealed inside a rectangular flow cell with optical readout windows. Oxygen concentration was varied by mixing air and nitrogen in defined ratios between 0 and 21% using a pair of digitally controlled mass flow controllers, using MKS instruments PR4000B controller to achieve dissolved oxygen levels between 0 and 258 µM. The air–nitrogen mixtures were bubbled into the TRIS buffer in a round-bottomed flask in an incubator and the buffer was circulated over the samples at 37 °C in the flow cell using a recirculating flow system described previously  was mixed with 25 µL of 33.3% aqueous suspension of calcium carbonate and 200 µL of 3.0% aqueous solution of sodium alginate. Finally, 100 µL of MES buffer with pH 6.1 was added and the whole mixture was quickly transferred into a rectangular mold made with microscope slides and 0.75 mm Teflon spacer. The release of calcium from the carbonate induced gelation of the alginate, which was allowed to proceed for 20 min. The resulting oxygen-sensing alginate hydrogel slab was carefully recovered and stored for 24 h in TRIS buffer (pH 7.2) with 10.0 mM calcium chloride at 4 °C before testing.−1) and 8.0 mg of N,N′-Methylenebisacrylamide (BIS) (Mol. Wt. 154.17 g mol−1) were dissolved in 406 µL of 50 mM TRIS buffer. The above mixture was mixed with 94 µL of nanoparticle suspension (1.56 mg of NPs). Next, 12.0 µL of 10.0 mM aqueous solution of ammonium persulfate (APS) and 2.0 µL of N,N,N′,N′-Tetramethyl ethylenediamine (TEMED) were added. Finally, the solution was bubbled with nitrogen for 30 s and quickly transferred into a rectangular sandwich mold with a 0.75 mm thick Teflon spacer. The above solution was placed in a nitrogen environment for 4 hours to allow crosslinking. The hydrogel was then washed in fresh 10 mM TRIS buffer containing 10.0 mM calcium chloride for 12 h and stored in the fresh TRIS buffer solution at 4 °C.In a typical synthesis of MPC hydrogel, 91.5 mg of MPC , 12.4 mg of acrylamide (mol. wt. 70.08 g mol−1), and 19.08 mg BIS (Mol. Wt. 154.17 g mol−1) were dissolved in 406 µL of 50 mM TRIS buffer by sonication. Next, 94 µL of nanoparticle suspension (1.56 mg of NPs) was added and mixed to obtain a homogenous mixture. To the above solution, 12.0 µL of 10.0 mM aqueous APS and 2.0 µL of TEMED were added, mixed and bubbled with nitrogen for 30 s. Finally, the reaction cocktail was transferred into a sandwich mold, as mentioned for other hydrogels, and placed under nitrogen environment for 4 h to allow complete crosslinking. After 4 h of reaction, the hydrogel was recovered, washed with fresh 10 mM TRIS buffer, and stored under TRIS buffer with 10 mM Calcium chloride at 4 °C.For preparation of HEMA hydrogel, 68.4 mg of 2-hydroxyethyl methacrylate (mol. wt. 130.14 g mol−1). Then, 94 µL of nanoparticle suspension (1.56 mg NPs) was added to the mixture and vortexed. Before polymerization, 12.0 µL of 10.0 mM aqueous APS and 2.0 µL of TEMED were added and nitrogen gas was bubbled, and the solution was transferred into a mold. Finally, the reaction mixture in the mold was placed under nitrogen for 4 h to allow crosslinking, washed with TRIS buffer containing 10.0 mM calcium chloride and stored in the same fresh buffer at 4 °C.First, 11.6 mg of BIS was dissolved in 295.75 µL of 50 mM TRIS buffer and mixed with 110.5 µL of 80% aqueous solution of 2-(Methacryloyloxy)ethyl]trimethylammonium chloride (mol. wt. 207.70 g molg using a homogenizer (see measurements and instrumentation). Next, 1.5 mL of isooctane containing 140 µL of TWEEN 85 was added while the above mixture was still mixing at 8000× g. Stirring was continued for 10 s to form a uniform emulsion, after which 4 mL of 10% w/w calcium chloride solution was added and the mixture was stirred for 15 s. Next, the obtained emulsion was transferred into a 500 mL round bottom flask and mixed at 300× g using a magnetic stirrer for 20 min to allow the crosslinking of alginate microparticles. The microemulsion was then centrifuged at 2000× g and washed twice with deionized water. Finally, particles were subjected to successive centrifugation cycles after mixing with 2% w/w solutions of PSS or PAH to form layers of alternating charge, as reported previously . SVK is the Stern–Volmer constant representing the slope of the linear relationship between the ratio 0τ/τ and [O2]. SVK was determined for each sample as the slope of the least-squares regression line for 0τ/τ plotted against oxygen.The lifetimes of the samples were recorded at 10 s interval as the oxygen leves were varied . Finally−1 glucose in TRIS buffer (pH 7.2) and “blank” TRIS buffer  were mixed at desired proportions and circulated through the flow cell containing the glucose sensors. Similarly, for the lactate-sensing experiment, a stock solution of 20 mg dL−1 lactate solution was used. The phosphorescence lifetime of the glucose sensors and lactate sensors were recorded in similar method as described for the oxygen sensitivity studies.To study the glucose and lactate-sensing characteristics, the hydrogel sensors were sealed inside a flow cell as mentioned above for the oxygen sensitivity analysis. Then, glucose or lactate concentration was systematically modulated to obtain the sensitivity data (see measurements and instrumentation section for details). For glucose sensing experiment, stock solutions of 400 mg dL2 supply at 37 °C. A cytotoxicity assay was performed according to the manufacturer’s protocol. Briefly, upon achieving 80% of cell confluence, fibroblasts were trypsinized and seeded in the 96 well-plates at 5000 cells per well seeding density in 100 µL of DMEM media . Non-encapsulated NPs were tested at concentration regimes similar to our standard microsphere concentrations dispersed within the hydrogel matrices (22 mg mL−1), which also equates to 4 mg mL−1 of microspheres under 10 uL of incubation volume. Therefore, exposure concentration regimes of non-encapsulated NPs, starting from the highest (4 mg mL−1) to its 1:10 dilutions were tested. For NPs encapsulated within microspheres, an appropriate exposure dose was first determined to avoid overwhelming cells with excess volume of microspheres coagulating at the bottom of the well. For this, we first calculated the estimated highest concentration of encapsulated NPs dispersed within the hydrogel matrix for any given sensor shape and dimension. Next, we designed our exposure dose regimes for encapsulated NPs starting from the highest (0.117 mg/mL) to 1:8 dilution and tested against the cells (more under NIH/3T3 (mouse embryonic fibroblasts) cells at passage 24 were grown in DMEM culture media supplemented with 10% FBS and 1X concentration of P/S antibiotics. Cell cultures were maintained in a humidified incubator with 5% COWe aimed to design a biocompatible, scalable, customizable, and sensitive oxygen sensor. We found that both PS and EC have been extensively used in the design of sensitive oxygen probes ,34. In a−1 (0.2% w/v). We further determined that dissolution of polymers, EC or PS in the 0.2% PdBP-THF solution did not lead to precipitation of the phosphor. We therefore used 1:10 PdBP:polymer in the precursor solution to achieve final dye concentration of 0.2% (w/v) and polymer concentration of 2.0% (w/v). The low concentration of PdBP was used to enhance aggregation-free encapsulation of the dye. Aggregation of phosphors is detrimental to oxygen-sensing systems, as it causes static quenching, formation of long-lifetime species, and consequently limits the dynamic lifetime range of a phosphor in response to oxygen  with a volume of 1.25 mm3. Based on our calculations, the highest dose of microparticles dispersed within cylindrical-shaped and strip-shaped sensor punches were estimated to be ~0.12 mg and ~0.03 mg, respectively. Therefore, we designed and tested the dose regimes for NIMs to cover the highest possible dose for each given form factor as well as additional lower doses .As depicted in As depicted in sv = 0.043 ± 0.006 µM−1) in free form and after encapsulating in microspheres and lowest overall cytotoxicity, we selected the EC-PF68 NPs for further evaluation within metabolite sensors intended as implanted devices. To demonstrate the utility of the EC-PF68 NPs in designing a sensitive metabolite sensor, we encapsulated EC-PF68, GOx (or LOx), and CAT in alginate hydrogel microparticles, as shown in Given the overall combination of high sensitivity (K−1 in TRIS buffer (−1 (1.1% dL mg−1). The response time to achieve 90% of the steady-state response following a step change in glucose concentration was estimated to be 20 min. This is slightly higher than the response time seen in FRET-based sensors with typical 4–10 min response in 0–200 mg dL−1 concentration range, and our previous observations based on HEMA based glucose sensors between 0 and 140 mg dL−1 glucose [−1. The glucose sensor response of 445% total increase in lifetime at 400 mg dL−1 relative to that at baseline (0 mg dL−1) is nearly 2× better than previously reported similar microdomain-based sensors based on direct dye loading with 25 diffusion control bilayers [−1 glucose [−1 glucose with 423% (47 to 197 µs) change in lifetime similar to our previous observation [−1, 106.2 mg dL−1, and 93.0 mg dL−1, respectively, based on a second degree polynomial fit between 0 and 150 mg dL−1 glucose  was calculated to be 13.7 mg dL−1 while the lactate sensitivity was 10.7 µs dL mg−1. Thus the sensing range, MDLC, and sensitivity are significantly improved by 29%, 47% and 223%, respectively, compared to our previous report where we directly trapped the phosphor directly in the alginate microdomains [To evaluate the generalizability of these systems, we further prepared and tested lactate sensors by encapsulating lactic acid oxidase in alginate microdomains along with EC-PF68 NPs and catalase enzyme. Here we employed 10 bilayers for effective diffusion control of lactate in the lactate-sensing microdomains in contrast to the 25 bilayers used in glucose sensors, based on observations from previous studies . As withodomains . The obssvK = 0.0026 µM−1), [block-vinylpyrrolidone) showed a dynamic lifetime range of 41 µs, [svK of 0.042 µM−1 (average of different hydrogel matrix and surfactant combined).The results in the current study compare favorably with other examples from recent published works. Fluorine-functionalized metal complexes in PS NPs were developed for enhanced oxygen sensitivity (6 µM−1),  nearly 56 µM−1),  the ethyf 41 µs,  and moref 41 µs,  platinumf 41 µs, . In cont70 for use in gaseous mixtures [Interestingly, most of the reported oxygen sensors that displayed trace oxygen sensitivity are known to be limited by their form factor and mostly used in gas phase. For example, a recently reported open source networkable oxygen sensor with a LoD of 26 ppm (gas phase) used 5,10,15,20-Tetrakis (pentafluorophenyl)-21H,23H-porphyrin palladium (II) coated on a glass disk as the sensing element . An oxygmixtures ,60,61. Omixtures . Metal omixtures . Thus, rsvK (average svK = 0.042 µM−1) values, wide phosphorescence lifetime range (208 µs) between 0 and 258 µM oxygen, and LoD of 0.0016 µM observed for EC NPs makes them one of the most sensitive systems capable of monitoring dissolved oxygen in aqueous media ever reported.In this study we have presented the design, optimization, and characterization of oxygen sensitive NPs encapsulating near-infrared phosphors; these are attractive for further incorporation into nano-in-micro (NIMs) metabolite sensors. Based on the high oxygen sensitivity, relatively low impact of matrix on sensitivity, and overall biocompatibility, we were able to successfully conclude that ethyl cellulose NPs coated with PF-68 (EC-PF68) present the best formulation of eight different combinations tested. The synthesized NPs are easily stored as aqueous suspension under ambient conditions, and they are not cytotoxic at realistic concentrations. Further, The high −1 glucose and <2 mM of lactate) [We further evaluated the potential use of EC-PF68 NPs in fabrication of NIMs-based glucose and lactate monitoring sensors, and tested with the physiologically relevant concentrations (70–140 mg dLlactate) ,65. The


Metabolic cage studies reveal that FA-treated mice have elevated energy expenditure by 11.5% during dark phases. Mechanistically, FA treatment increases the expression of rate-limiting enzymes of fatty acid oxidation and ketone body biosynthesis CPT1A, ACOX1 and HMGCS2, which are the peroxisome proliferator-activated receptors α (PPARα) targets in liver. In conclusion, FA could effectively prevent HFD-induced NAFLD possibly by activating PPARα to increase energy expenditure and decrease the accumulation of triacylglycerol in the liver.There is a consensus that ferulic acid (FA), the most prominent phenolic acid in whole grains, displays a protective effect in non-alcoholic fatty liver disease (NAFLD), though its underlying mechanism not fully elucidated. This study aimed to investigate the protective effect of FA on high-fat diet (HFD)-induced NAFLD in mice and its potential mechanism. C57BL/6 mice were divided into the control diet (CON) group, the HFD group, and the treatment (HFD+FA) group, fed with an HFD and FA (100 mg/kg/day) by oral gavage for 12 weeks. Hematoxylin and eosin (H&E) staining and Oil Red O staining were used to evaluate liver tissue pathological changes and lipid accumulation respectively. It was demonstrated that FA supplementation prevented HFD-induced NAFLD, which was evidenced by the decreased accumulation of lipid and hepatic steatosis in the HFD+FA group. Specifically, FA supplementation decreased hepatic triacylglycerol (TG) content by 33.5% ( Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide, with a global prevalence of approximately 25% . It is cPeroxisome proliferator-activated receptors (PPARs) regulate energy metabolism and are therefore therapeutic targets in NAFLD . Among tIn a state of fasting, metabolic substrates stored in white adipose tissue are released into the circulation as glycerol and free fatty acidsand transported into the liver . The livFerulic acid  is one of the main natural phenolic acids in cereals including rice , rye 1313, wheatIn the present study, we investigated the mechanism by which FA prevents high-fat-diet-induced NAFLD. This work will help to improve the understanding of the potential mechanism underlying the protective effect of FA on NAFLD.Ferulic acid  and the β-Hydroxybutyric Acid Content Assay Kit were purchased from Solarbio Science & Technology co., LTD . Primers  used in w/v) was orally administered to the HFD+FA mice via a gastric tube, while the CON group and the HFD group also received an equal volume of the vehicle, sodium carboxymethylcellulose. Body weight and food intake were recorded daily. At designated experimental endpoints, all mice were anesthetized with isoflurane and cervical dislocation after a 12 h fast. Serum and tissue samples were then collected by either snap-freezing in liquid nitrogen and storing at −80 °C or directly storing in 4% paraformaldehyde for histological analysis. All animal experimental procedures were conducted following the Guide for the Care and Use of Laboratory Animals: Eighth Edition (ISBN-10: 0-309-15396-4). We have complied with all relevant ethical regulations for animal experiments. All the protocols were approved by the Faculty Animal Policy and Welfare Committee of Northwest A & F University, China (Permission ID: 20200528-010), and therefore the animal experiments were carried out in accordance with all animal care regulations, guidelines, and standards.Five-week-old male C57BL/6J mice were obtained from the Experimental Animal Center of Xi’an Jiaotong University . The mice were fed a normal chow diet for an acclimatization period of 1 week after their arrival. A schematic diagram of mice treatment is shown in −1 body weight per day for mice was equivalent to 500 mg FA per day for a mean human weight of 60 kg, according to the formula for dose translation based on body surface area: mouse equivalent dose (mg kg−1) = human dose (mg kg−1) × (Km-adult/Km-mouse), Km factor of 3 and 37 for adult mouse (0.02 kg) and adult human (60 kg), respectively [The dosage of FA used in this study was calculated based on pilot experiments and the reference at which no toxicity was observed in humans ,23. The ectively .2), carbon dioxide production (VCO2), and respiratory exchange ratio (RER) were measured every five minutes. The RER was derived from the ratio of VCO2 to VO2, and energy expenditure was determined as (3.815 + 1.232 × RER) × VO2 and expressed as kcal h−1. Total body weight was used as the covariate in the analysis.Mice were individually housed in metabolic cages  at room temperature (22 ± 2 °C) and allowed to acclimate for 24 h. After acclimatization, mice were gavaged with FA (100 mg/kg) every morning for 3 days. Oxygen consumption . Mice were gavaged with glucose (2.0 g/(kg of bw)), the glucose levels of tail vein blood samples were measured at 0, 15, 30, 45, 60, 90, and 120 min using a glucose analyzer . For the insulin tolerance test (ITT), mice were fasted for 6 h and then administered insulin (0.75 units/(kg of bw)) by intraperitoneal injection. The glucose levels of tail vein blood samples were measured at 0, 15, 30, 45, 60, 90, and 120 min after insulin load.Serum levels of 3-hydroxybutyric acid were quantified using the β-Hydroxybutyric Acid Content Assay Kit following the manufacturer’s protocol. The hepatic β-hydroxybutyric levels were determined using UPLC-MS/MS. The hepatic TG levels were determined using an automatic biochemical analyzer  and normalized to those of the protein concentrations in the initial homogenate according to the manufacturer’s instructions. The murine liver tissues were sequentially fixed with 4% paraformaldehyde, dehydrated with gradient ethanol, embedded in paraffin, and sectioned at 5 μm with a microtome . Afterwards, the tissue sections were dried overnight at 37 °C. For H&E staining, the sections were heated at 60 °C for 1 h, deparaffinized, rehydrated, and then stained with hematoxylin and eosin . Subsequently, the stained sections were dehydrated in gradient ethanol and xylene, sealed with neutral balsam, and air-dried at room temperature. For Oil Red O Staining, the samples were rinsed with PBS and fixed in 10% buffered formalin, then stained with Oil Red O (0.5 g in 100 mL of isopropanol) for 60 min. After discarding the staining solution, isopropanol was added to the samples to elute the retained dyes. After mounting, the sections were visualized and photographed using an optical microscope with camera  at a 100× or 400× magnification. ® Premix Ex Taq™ II , 0.8 μL specific forward/reverse primer (10 μM), 1 μL cDNA, and ddH2O to a final volume of 20 μL. The quantitative PCR was performed using the following conditions: 95 °C for 5 min, 40 cycles of 95 °C for 5 s, and the optimized annealing temperature for 30 s. There were 6 samples in each group, and the reaction of each sample was performed in duplicate. Total RNA was extracted from the murine liver tissue using AG RNAex Pro Reagent  following the manufacturer’s instructions. For quantitative real-time PCR (qRT-PCR) analysis, the first-strand cDNA was obtained using an Evo M-MLV RT Kit with gDNA Clean for qPCR , and the mRNAs were quantified using the Bio-Rad CFX96 Touch™ Real Time PCR Detection System  with β-actin as an endogenous control. The qRT-PCR reaction consisted of 10 μL 2 × SYBRg for 5 min. The supernatants were collected and the protein concentrations measured using a BCA Protein Assay Kit . Protein lysates were analyzed at a concentration of 40 μg per sample and separated with SDS-PAGE before transferring to the polyvinylidene fluoride (PVDF) membrane . The membrane was blocked with 5% non-fat milk, followed by incubation with antibodies against β-actin, ACOX1, CPT1α, HMGCS2, or PPARα at 4 °C overnight. The membrane was then incubated with HRP-conjugated secondary antibody (1:5000) at 37 °C for 2 h and labeled in the dark with a chemiluminescence ECL kit  according to the manufacturer’s instructions. Finally, the protein bands were visualized with a Bio-Rad Chemidoc  and the optical density was analyzed with the software ImageJ 1.53. The protein expression was normalized using β-actin  as an internal control.Liver samples were lysed with RIPA buffer  containing 1 mM PMSF . Subsequently, the lysate was centrifuged at 12,000× t-test and one-way analysis of variance (ANOVA), followed by Tukey’s post hoc comparison test. Statistical analyses and figures were performed by Prism v8.2.1  or the R package (v3.5.2). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.All numerical values are presented as mean ± SEM. Statistical analyses were conducted using an unpaired p < 0.01)  D.p < 0.05) compared with the HFD group (p < 0.01) compared with the HFD group  typically exceeds the rate of fatty acid output (including secretion of very-low-density lipoprotein and fatty acid oxidation) . TherefoFD group , indicatFatty acid β-oxidation in the liver is accompanied by ketone body biosynthesis . KetonesOf note, the mice treated with FA exhibited stationary RER, indicating that both lipids and carbohydrates used as the energy substrate are elevated. The same phenomenon was also observed in mice treated with a PPARα agonist . The curOverall, the present study clearly showed that FA, the most represented phenolic acid in whole grains, was an effective compound to prevent NAFLD development in vivo. The mechanism by which the FA activates PPARα to prevent NAFLD involves the following: Firstly, FA supplementation decreased hepatic TG accumulation via promoting fatty acid β-oxidation and ketone body biosynthesis to increase fatty acid expenditure. Secondly, FA supplementation increased energy expenditure via increased lipids used as the energy substrate. Therefore, these observations may provide a promising candidate for the prevention of NAFLD, and FA can be developed as functional foods or therapeutic medicines.


QSNCs exhibited conical and parallelepiped shapes. An increase of the QSNC concentration, from 0 to 5%, improved the film’s tensile strength from 6.5 to 16.5 MPa, but at 7.5%, it decreased to 11.85 MPa. Adequate exfoliation of QSNCs in the starch matrix also decreased the water vapor permeability (~17%) up to a 5% concentration. At 5.0% and 7.5% concentrations, the films increased in roughness, water contact angle, and opacity, whereas the brightness decreased. Furthermore, at these concentrations, the film’s hydrophilic nature changed . The SNC addition increased the film opacity without causing major changes in color. Other film properties, such as thickness, moisture content and solubility, were not affected by the QSNC concentration. The DSC  results indicated that greater QSNC concentrations increased the second glass transition temperature (related to the biopolymer-rich phase) and the melting enthalpy. However, the film’s thermal stability was not altered by the QSNC addition. These findings contribute to overcoming the starch-based films’ limitations through the development of nanocomposite materials for future food packaging applications.Quinoa starch nanocrystals (QSNCs), obtained by acid hydrolysis, were used as a reinforcing filler in cassava starch films. The influence of QSNC concentrations (0, 2.5, 5.0, 7.5 and 10%,  Manihot esculenta) is an important source of relatively cheap starch, possessing excellent film-forming properties [Currently, the development and applications of biopolymer-based biodegradable films are one of the main interesting trends in food and materials sciences for the replacement of synthetic polymers, which are derived from the petrochemicals used in packaging ,2. Amongoperties ,4.Starch-based films present desirable characteristics for food packaging applications because they are homogeneous, odorless, tasteless, colorless, and non-toxic, and are semipermeable to oxygen, carbon dioxide and flavor components . DespiteThe interest in this type of nanomaterial is growing because of their abundance, biodegradability, biocompatibility and particularly for coming from a renewable resource . The mosSeveral authors observed that the addition of SNCs from different sources, such as waxy maize, rice, taro and potato, into starch -film matrices improved the films’ mechanical properties ,16,17, aChenopodium quinoa Willd.). Quinoa is an Andean grain crop with 53.5–69.2% of its dry weight as starch, its main storage compound [In this sense, the A-type starches have been associated with SNCs with platelet morphology and greater thermal stability , which icompound , making Quinoa starch (QS) presents with an A-type crystalline pattern, a small granule size (0.5–3 μm) with polygonal shapes, and low amylose content (7–26%) and gelatinization temperatures (54–78 °C) , which aCurrently, there is limited research on the application of SNCs from unconventional sources as a reinforcing filler in starch-based films; although potato, rice and taro starches have been studied ,17,19, tQuinoa grains of the “Real” variety originally from Bolivia were bought in Pirassununga . Commercial cassava starch (CS) was acquired from H. L. do Brasil Indústria e Comercio de Produtos Alimentícios LTDA in São Paulo, Brazil . The cassava starch contained 12.31 ± 0.04% moisture content (w.b.), 0.39 ± 0.02% lipids (d.b.), 0.16 ± 0.00% ash (d.b.), 0.26 ± 0.02% protein (d.b.), and 16.90 ± 0.18% (starch basis) apparent amylose content; these results were obtained by employing the AOAC International standard methods .Quinoa’s starch extraction yield, chemical composition and apparent amylose content have been previously reported in detail by Velásquez-Castillo et al. . The reaw/w), neutralized with a citric acid solution (1 M), washed with distilled water and dried. The isolated QS was ground, then sieved through a 100-mesh size and kept at room temperature.The QS was isolated by an alkaline method, as detailed by Velásquez-Castillo et al. . Briefly2SO4 solution. The resulting suspension was incubated at 35 °C with stirring at 200 rpm for 5 days. The final suspension was washed with distilled water using successive centrifugations  until neutrality, and then the precipitate was freeze-dried  to obtain powdered QSNC.The QSNCs were prepared by acid hydrolysis of QS ,28. The The CS and QS morphologies were analyzed by scanning electron microscopy (SEM)  at 15 kV, and the QSNC were studied by field emission gun scanning electron microscopy (SEM-FEG)  at 10 kV, allowing magnifications of 3000× and 50,000×, respectively . The QSNThe CS, QS and QSNC X-ray diffraction patterns were obtained employing a diffractometer  with Cu Kα radiation (λ = 1.54056 Å) at 40 kV and 15 mA. Samples (30 mm × 40 mm) were analyzed from 4 to 40° (2θ) at 2°/min . The cryw/w) were prepared in distilled water and were then heated at 90 °C for 30 min under mechanical stirring at 300 rpm to allow starch gelatinization. The plasticizer, glycerol at 25% , was added, and the film-forming dispersions were stirred for 15 min and cooled to 50 °C. QSNC, previously dispersed in distilled water using an Ultraturrax  at 10,000 rpm for 10 min, was added to the CS dispersions at 0, 2.5, 5.0, 7.5 and 10%  concentrations under stirring at 300 rpm for 15 min, producing film-forming dispersions (FFD).The films were prepared by the casting technique according to Li et al. , with soThe FFDs (about 48 g) were spread evenly over Petri dishes (14 cm diameter) and dried in a forced-air circulation oven  at 30 °C for 21 h. The films were peeled off from the supports and conditioned in desiccators containing NaBr-saturated solutions (58% RH) at 25 °C for at least 5 days before the characterizations were made. For the microstructure and FTIR analyses, the samples were conditioned over silica gel at 25 °C for at least 7 days.Film thickness was measured using a digital micrometer  with an accuracy of 0.001 mm. Thickness values were considered as the mean of ten measurements taken at random points ,18. The Film solubility in water (SW) was determined gravimetrically . Previou2), P1 is the vapor pressure of pure water at 25 °C (3.166 kPa), and P2 is the vapor pressure of the atmosphere with silica gel (0 kPa).Film water vapor permeability (WVP) was determined with the ASTM E96/E96M method . CirculaThe film’s color was determined using a colorimeter  in the reflectance mode with a D65 illuminant, 10° angle and 30 mm aperture. The CIELab standard was used to measure the film’s luminosity (L*), which ranged from 0  to 100  with a* varying from green (−a) to red (+a) and b* from blue (−b) to yellow (+b). The films were placed onto white and black standard plates , and then the total color difference (∆E*) was calculated using Equation (2).b/Yw) was determined as the ratio between the film opacity when placed onto black (Yb) and white (Yw) standard plates [The film opacity was determined with the HunterLab method in reflectance mode, employing the same colorimeter as for the color measurement. The opacity  and the root mean square roughness (Rq: root mean square average of the height deviations taken from the mean data plane) were calculated using the microscope software .The film’s microstructure (air-side surface) was analyzed using an atomic force microscope  operating in a semi-contact mode  . DiffereFilm gloss was measured according to the ASTM D2457 standard  using a The film water contact angle (WCA) was measured using an optical tensiometer , as indicated in the ASTM D7334 standard method . The samFilm X-ray diffraction patterns were obtained as described in Film mechanical properties were determined by the uniaxial tensile tests according to ASTM D882-10 , using ag), the melting temperature (Tm), and the melting enthalpy (ΔH) were determined directly from the heat-flow curves using the Universal Analysis V1.7F software (TA Instruments).Film thermal properties were analyzed using a differential scanning calorimeter . Furthermore, 10 mg samples were placed in an aluminum capsule, hermetically sealed, and analyzed from −150 to 250 °C at 10 °C/min . The glaThe film’s thermal stability was analyzed using a thermogravimetric analyzer . Furthermore, 10 mg samples were placed in the sample holder and heated from 30 to 600 °C at 5 °C/min under an inert air atmosphere (20 mL/min) .−1; resolution: 4 cm−1; 32 scans per sample [The film’s chemical structure was studied using a Fourier transform infrared (FTIR) spectrometer  with a universal attenuator total reflectance accessory. The spectral range was from 600–4000 cmAll tests were performed in triplicate. The results were expressed as mean ± standard deviation and evaluated by the analysis of variance. The Statistica software  was used to analyze the means with Tukey’s test at a 5% significance level.The QS granules presented spherical shapes in a very low dimension and were disposed of in aggregates structured by a protein matrix a. AraujoConversely, the QSNCs appeared as individual parallelepiped and conical structures, respectively c, which The AFM micrographs of QSNCs e,f allowQS, CS and QSNC X-ray diffraction (XRD) patterns presented broad peaks with high intensity at (2θ) 15°, 17°, 18° and 23° a, typicaThe crystallinity indices from CS, QS and QSNC were 28.6, 26.6 and 34.5%, respectively. These values were in accordance with the literature for the starches ,39 and SFilms up to 7.5% of QSNC were easily handled and removed from the Petri plates and presented macroscopic homogeneity . The filThe film thickness was 0.103 mm on average, with no significant changes due to the different QSNC concentrations , indicatThe film MC was approximately 11% and not significantly affected by the QSNC concentration . This beFilm SW remained around 24% and was also not affected by the QSNC concentration . SimilarOnly the CS film with 5% QSNCs presented a significantly lower WVP value than the pure film . This caDespite the significant effect in L* , all filCondés et al.  also repp < 0.05) from 0.5 to 3.3 because of the increase of the QSNC concentration from 0 to 7.5%  increased from 85–102 to 205–247 nm, and the root mean square roughness (Rq) increased from 108 to 261–309 nm , R2 = 0.82) and Rq (Equation (4), R2 = 0.86), with almost the same tendency (similar slopes). This was in accordance with the observations for other nanoparticles, such as Laponite© [Considering the gloss values determined at 60° , all filaponite©  in starca (Equation (5), R2 = 0.88) and Rq (Equation (6), R2 = 0.89).The increase in the QSNC concentration raised the film surface hydrophobicity; therefore, the droplets were smaller, had a larger base for the lower QSNC concentrations and were higher with a shorter base for the higher QSNC concentrations . The filThe film-specific surface increased as a consequence of its roughness, contributing to raising the adhesion force between the water droplet and the film surface. Condés et al.  and Dai Film X-ray diffractograms were characterized by a wide peak around 20° b, which The CS film tensile strength (TS) and Young’s modulus (M) increased around 2.5 times when the QSNC concentration increased from 0 to 5% , and theNevertheless, when the QSNC concentration increased to 7.5%, the exfoliation quality was lost due to the eventual SNC agglomeration that caused a loss of stress transfer effectiveness, as observed by Dai et al.  working gG (~−71.4 °C) was not affected by the QSNCs and was attributed to a glycerol-rich phase, while the second one, TgS, associated with the biopolymer-rich phase [The film thermal curves, determined by DSC a, evidench phase , increasch phase ,19. Simich phase .m), which is linked to starch degradation, was not affected by the QSNC addition, remaining at around 213 °C (m increased in starch films reinforced with waxy maize SNC (5%), potato SNC (0.1–0.3%) and taro SNC (2.5−10%) due to SNC-film matrix interactions, which increased film crystallinity. Thus, the behavior observed in this study could be related to QSNCs’ characteristic physical properties, particularly regarding their low crystallinity (~35%), which in the studied concentrations, did not influence Tm. In contrast, the QSNC addition did increase film enthalpy from 56–61 to 161–147 J/g when the concentration increased from 0–2.5 to 5–7.5% , 2928 cm−1 (C-H), 1645 cm−1 (O-H), 1015 cm−1 (C-O-C), and 993 cm−1 (C-O) [−1 slightly decreased after the QSNC addition, possibly related to a diminution of the available hydroxyl groups due to intermolecular hydrogen bonding among the QSNC hydroxyl groups and the starch matrix [−1, which is broadly associated with ordered structures [Film FTIR spectra  showed t−1 (C-O) . These b−1 (C-O) . Neverthh matrix . Moreoveructures . This coructures .w/w of QSNC was the best concentration regarding the tensile strength and Young’s modulus. These properties were lower for the QSNC concentrations above or below 5%. At 5% of QSNC, the tensile strength increased up to 154% in relation to the films without nanoparticles. Moreover, CS films with 5% of QSNC presented a 17% lower WVP. The reduction of the tensile strength and Young’s modulus above 5% of QSNC was due to a loss in the nanoparticle dispersion quality.The physical and structural properties of the cassava starch films showed that the QSNC application as a reinforcing filler was viable and relevant, mainly in relation to the mechanical (tensile strength and Young’s modulus) and barrier (WVP) properties of the material without degradation of its other physical properties. In this study, 5% The QSNC concentration also affected the film surface roughness, gloss, opacity and water contact angle. The effect of QSNCs on gloss and the water contact angle was due to the alteration caused by nanoparticles on air-side film roughness. The FTIR analysis suggested the formation of hydrogen bonding without forming any chemical bonding between QSNCs and cassava starch as a function of the QSNC concentration, which was indicated by its solubility in water and thermal properties. This work evidenced that QSNCs acted as a reinforcement of CS films, indicating their potential for the development of nanocomposite starch-based films, which can be used in food packaging applications.


In vivo analyses confirmed that ECM1 overexpression was able to enhance EMT induction and CRC tumor progression. In conclusion, ECM1 influences CRC development and progression in an oncogenic manner, and regulates CRC metastasis and EMT processes via the PI3K/AKT/GSK3β/Snail signaling axis.In prior reports, extracellular matrix protein 1 (ECM1) upregulation has been reported in colorectal cancer (CRC) patient tumor tissues, and has been suggested to be related to the metastatic progression of CRC, although the underlying mechanisms have yet to be clarified. In this study, we found that ECM1 was overexpressed in both CRC tissues and cell lines. Upregulation of ECM1 was correlated with tumor size, lymph node status and TNM stage in CRC patients. Knocking down ECM1 suppressed CRC cell growth, migration and invasion, in addition to reducing the expression of Vimentin and increasing E-cadherin expression. The overexpression of ECM1, in contrast, yielded the opposite phenotypic outcomes while also promoting the expression of p-AKT, p-GSK3β, and Snail, which were downregulated when ECM1 was knocked down. Treatment with LY294002 and 740 Y-P reversed the impact upregulation and downregulation of ECM1 on CRC cell metastasis and associated EMT induction.  One of the most prevalent gastrointestinal malignancies in the world is colorectal cancer (CRC) . While 5The secretory glycoprotein, extracellular matrix protein 1 (ECM1), is associated with physiological processes including angiogenesis, the proliferation of epithelial cells, and skin differentiation, in addition to playing a role in tumor progression , 4. Recein vitro and in vivo assays after the knockdown or overexpression of ECM1. We additionally explore the mechanisms whereby ECM1 regulates the growth, invasion, and migration of CRC cells and associated metastatic changes. Through this approach, we found that the regulation of the occurrence and progression of CRC by ECM1 was mediated through the PI3K/AKT/GSK3β/Snail signaling pathway.In the present study, we confirmed that ECM1 was overexpressed in CRC cancer tissues and cell lines. To further clarify the role of ECM1 in CRC, we performed www.oncomine.org) was used to compare ECM1 expression levels between CRC tissues and normal tissues.Oncomine , with all contributors having presented the letter of informed consent to participate.Formalin-fixed paraffin-embedded tissues were sliced to prepare 3 μm thick sections. Antigen retrieval was conducted using citrate buffer  based on the protocols included in an IHC staining kit . Slides were probed overnight with primary anti-ECM1 at 4°C, followed by incubation with an appropriate secondary antibody at room temperature for 30 min. In negative control sections, PBS was employed in lieu of primary antibody. As per a previous study , IHC sco2 incubator at 37°C.NCM460, HCT116, HCT15, HT29, SW480, 293T, and SW620 were purchased from BeNa Culture Collection . NCM460 and 293T cells were grown in HG media (Gibco), while SW620, HCT116, HT29, SW480, and HCT15 cells were grown in RPMI-1640 (Gibco) including penicillin/streptomycin (Gibco) and 10% FBS (Gibco). All cells were cultured in a humidified 5% CO5/well) and grown to 40-60% confluence, at which time Lipofectamine 2000 (Invitrogen) was used to transfect cells with appropriate siRNAs (Genepharma), followed by incubation for 6 h in serum-free media, after which media was exchanged for media containing 10% FBS. Following an additional 24 h incubation, qPCR was performed to assess ECM1 knockdown efficiency, with Western blotting and cell functional assays being performed at 48 h post-transfection.Cells were added to 6-well plates . After 72 h, supernatants were collected, centrifuged for 20 min at 3000 rpm, filtered, centrifuged at 4°C for 20 min at 5000 rmp and concentrated. The resultant lentiviral concentrates were then used to infect cells, generating cell lines expressing ECM1 or the empty vector control.® qPCR RT Kit . Subsequent qPCR reactions were performed employing SYBR® Green Realtime PCR Master Mix  with an Applied Biosystems 7300Plus Real-Time PCR Instrument . The 2-ΔΔCt approach was utilized for the assessment of relative gene expression, with analyses being conducted in triplicate. Utilized primers are as follows: β-actin-F: 5’-ACCCCGTGCTGCACCGAG-3’, β-actin-R: 5’-TCCCGGCCAGCCAGGTCCA-3’; ECM1-F: 5’-CCTGGGCTGATCCACAACAT-3’; ECM1-R:5’-TTGGCGTTCTCAGTGTCTCC-3’.The Tsingzol RNA Extraction Reagent  was utilized to extract RNA from cells and tissue samples, after which cDNA was produced with a ReverTra Acevia 10% SDS-PAGE and transferred to PVDF membranes (Millipore) that were then blocked for 2 h with 5% non-fat milk at room temperature, followed by overnight incubation with primary antibodies diluted in TBST containing 5% non-fat milk at 4°C. Blots were then incubated at room temperature with HRP-conjugated anti-rabbit or anti-mouse IgG , after which a chemiluminescent imaging system was used for protein band detection. Primary antibodies included antibodies specific for the following, all of which were diluted at 1:1000 before use: ECM1 , E-cadherin , Vimentin , Snail , AKT , p-AKT (Ser473) , GSK3β , p-GSK3β (Ser9) , GAPDH . Image J was used for quantitative analyses.RIPA Buffer  containing 1% PMSF  was used to extract protein from cell and tissue samples, after which a BCA Kit (Beyotime) was employed to measure protein concentrations. Equal protein amounts from individual samples  were subsequently separated CCK-8 and colony formation assays were used to appraise cellular proliferation. Briefly, cells were added to 96-well plates (5000 cells/well in 100 uL) and incubated until adherent. CCK-8 reagent  was then added to each well after 0, 24, 48, or 72 h, followed by an additional 2 h incubation at 37°C. Absorbance at 450 nm was subsequently analyzed with a microplate reader .Cells were added to 6-well plates (1000/well) and cultured until colony formation was evident, at which time 4% paraformaldehyde was used to fix colonies which were then stained for 1 min with 0.2% crystal violet at ambient temperature. Colonies were then counted using ImageJ.5/well) and cultivated for 24 h, at which time they were harvested using trypsin, rinsed with PBS, and fixed for 24 h with 70% ethanol at 4°C. Cells were subsequently stained with an AnnexinV/PI double staining kit  based on provided directions, followed by analysis via flow cytometry . Apoptotic cell death was assessed by plating cells under the same conditions, and then harvesting and rinsing these cells three times using PBS prior to resuspension in 100 uL of binding buffer. Cells were next stained with 5 uL of Annexin V-FITC and 10 uL of PI for 15 min on ice, after which 400 uL of binding buffer was added. Cells were analyzed within 1 h via flow cytometry .To assess cell cycle progression, cells were added to 6-well plates (3× 105 in 200 uL) were added to the upper chamber of each well, while 600 uL of RPMI-1640 contaning 20%FBS was added to the lower chamber. Following culture for 24 h, cells were fixed with 4% paraformaldehyde for 30 min, stained for 1 min with crystal violet, and non-migratory/invasive cells were eliminated with a cotton swab. Those cells bound to the lower chamber surface were then imaged via microscopy, with three random fields of view per sample being assessed.Transwell polycarbonate inserts (8 μm pore size) that were or were not coated with Matrigel  were used with 24-well plates to assess cellular migration and invasion. Cells (2x106/well) and were grown until 90% confluent, at which time a 200 uL pipette tip was used to generate a scratch wound in the monolayer surface. Cells were then rinsed with PBS and cultured in RPMI-1640 containing 1%FBS for 24 h, with the rate of wound healing (%) being determined by measuring the area between the two sides of the wound using ImageJ. Wound healing rate = (area at 0 h - area at 24 h)/area at 0 h.Cells were added to 6-well plates (1×106 cells/50ul). On day 25 post-inoculation, mice were anesthetized and humanely euthanized, at which time tumors were weighed and collected for downstream analyses.Male BALB/c nude mice  were housed in a temperature-controlled facility with a natural day/night cycle and free access to water and food. Laboratory Animal Welfare and Ethics Committee of Chongqing University approved all animal studies, which were performed in a manner consistent with the Guide for the Care and Use of Laboratory Animals. A xenograft tumor model was established by subcutaneously injecting mice in the right flank with HCT116-NC or HCT116-ECM1 cells . *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.Statistical analyses were performed using SPSS 26.0, GraphPad Prism 9.0, and ImageJ. Differences were compared between groups using two-tailed t-tests, while the relationships between ECM1 expression and clinicopathological characteristics were evaluated P<0.001), lymph node metastasis (P<0.001), and tumor size (P<0.05) . ECM1 upregulation was associated with a significant increase in both tumor volume and weight  and The Talents project of Chongqing, China (CQYC202103063).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aims to prepare and characterize nanosystems based on porphyrin grafted carbon nanotubes (SWCNTs), to understand some aspects of the mechanisms behind the SDT phenomenon. Three different porphyrins have been covalently linked to SWCNTs using either Diels–Alder or 1,3-dipolar cycloadditions. ROS production and cell viability have been evaluated upon ultrasound irradiation. Despite the low porphyrin content linked on the SWCNT, these systems have shown high ROS production and high tumour-cell-killing ability. The existence of a PET (photoinduced electron transfer)-like process would appear to be able to explain these observations. Moreover, the demonstrated ability to absorb light limits the impact of side effects due to light-excitation.Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given molecule  by inertial acoustic cavitation that leads to cell death  via the production of reactive oxygen species (ROS).Sonodynamic therapy (SDT) is an innovative anticancer approach, based on the excitation of a given molecule  by inertial acoustic cavitation that leads to cell death  Sonoluminescence is a process, invoked in a controversial way,5,6 in which gaseous species dissolved in solution are excited by acoustic cavitation and become able to emit light. The light emitted would excite sonosensitizer molecules  which, in turn, induces the generation of ROS . On the other hand, the sonosensitizer excitation could be directly produced by the high punctual temperature and pressure conditions induced by cavitation, following a thermal energy transfer mechanism. Moreover, direct radical production from water or from the sonosensitizer itself could also be implied.7–9 Porphyrin derivatives, which are non-toxic to cell culture per se10 are considered as the first-generation sensitizers and extensively used as model compounds in photo- and sono-dynamic treatment.11,12 However, in the clinical practice prolonged skin photosensitivity after systemic administration of porphyrins is observed.13 This side-effect represents one of the major shortcoming in the clinical use of such sensitizers.14 To overcome this problem it is possible to select sonosensitizer with different structure, or to select molecules able to be excited only by US. Another strategy envisages the enhancement of the sonosensitizer ability to generate ROS. This approach allows to reduce the dose and, consequently, the sonosensitizer concentration in the skin. Furthermore, the sonosensitizer may also be made tissue selective by conjugating it to a specific vector or including it in targeted nanoparticles. To conjugate sonosensitizer to vector, more than one strategy can be simultaneously followed. Single walled carbon nanotubes (SWCNT), are one of the most promising materials in nanomedicine15,16 and they represent an ideal model to build a new kind of porphyrin based sonosensitizer (porphyrin–SWCNT conjugated). SWCNT are known for their use in drug delivery and targeted drug delivery,17,18 but they are also known for their peculiar dimensional features  and electronic properties.19–21 Three main advantages are expected from the porphyrin–SWCNT nanohybrid: (i) as described by Baskaran and co-workers,22 the stiffness and micrometric size of SWCNT bundle favour the inertial acoustic cavitation to occur in the proximity of the carbon surface where the sonosensitizing agent is linked, (ii) the exceptional electron mobility of SWCNT23 assists the electron transfer between covalently bonded molecules and the nanotube, imparting a US-harvesting antennae behaviour20,22,24–26 and (iii) SWCNT allows a facile chemical grafting16 of porphyrin derivatives onto the carbon surface and allow further functionalization with target agents.15,17,27,28 In this study three different porphyrin–SWCNT nanohybrids were synthesized and studied in in vitro sonodynamic applications.Sonodynamic therapy (SDT) was introduced as an innovative anticancer approach in 1989.ad hoc tailored porphyrin derivatives. The three nanohybrid systems , obtained by grafting these porphyrins onto SWCNT surface -porphyrin with α-Boc-lysine t-butylester in presence of ethyl-diisopropylcarbodiimide (EDC) and 4-N,N-dimethylaminopyridine (DMAP).To assess if the ability in amplifying/facilitating the inertial cavitation conditions and the electron transfer phenomenon from porphyrin to SWCNT may increase toxic effect on cancer cells, we conjugated the SWCNT surface with three  surface , differ Porphyrins 1 and 3 expose furanyl terminal groups, which are appropriate for Diels–Alder (DA) cycloadditions. Porphyrin 2 was synthetized to employ the α-aminocarboxylic moiety as precursor of azomethine ylides, which is exploitable in 1,3-dipolar cycloadditions. All grafting reactions with porphyrin 1, 2 and 3, were performed by mono-modal microwave enhanced cycloaddition. The functionalization degree of these three hybrid materials was assessed by thermogravimetric analysis  accounts for a minor weight loss (<3%). The weight loss at this temperature (270 °C) was used as reference point (offset) to calculate the amount of porphyrin of each conjugate. The amount of porphyrin was quantified by subtracting the offset of each curve at 270 °C from the weight loss at 700 °C, in which the thermal degradation of porphyrin is completed. The percentage of porphyrin per mass unit of conjugate (SWCNT + porphyrin) introduced by the functionalization process in SWCNT–1, SWCNT–2 and SWCNT–3 was 6.7% w/w, 8.0% w/w and 7.0% w/w, respectively. To obtain the functionalization degree  the wt% were converted by using the molar mass of each porphyrin. The functionalization degree of SWCNT–1, SWCNT–2, and SWCNT–3 reported as μmol of porphyrin per mg of sample was calculated as follows: functionalization degree = (Δ700–270 °C (mass loss%)/MW porphyrin) × 103. This result  and 160–270 °C (region II) the desorption of molecular water and traces of solvent deriving from the synthesis (s result  evidencesn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000] ammonium salt) under mild conditions.31 The attachment of PEG was confirmed by thermogravimetric analysis  chains by using mPEG-DSPE  or SWCNT–PEG (negative control) and exposed to US irradiation. The results were compared to those obtained employing light irradiation , following previously published procedures.11,21,24 Cell proliferation at 24 and 48 hours after each treatment was assessed  generation by porphyrin loaded SWCNT hybrid was assessed by employing, terephthalate assay.35 US irradiated porphyrin promotes ROS production with consequent generation of hydroxyl radicals which convert terephthalate into its fluorescent derivative 2-hydroxyterephthalate (TA-OH). SWCNT–1 in DMSO : water (1 : 3) suspension was subjected to US irradiation in the dark. The experiments were carried out under two different US irradiation conditions: 1.866 MHz and 22 kHz. The former was selected in order to match the experimental condition used in cell treatments and the latter to test our system in well-studied ROS production conditions.2 ROS production of SWCNT–1 was compared with pristine tube (SWCNT) under the same experimental conditions adopted in cell experiments. To take into account ROS production via water sonolysis, an aqueous solution of TA was irradiated under the same conditions without SWCNT (blank + US). All the results were also compared to a solution not subjected to sonication (blank), to consider TA autoxidation. Under both US frequencies, the ROS generated by SWCNT–1 were significantly higher than those observed with pristine SWCNT (p < 0.05). Notably, pristine SWCNT did not induce ROS production more than blank solution, indicating that the radical generation by our hybrid nanomaterial relays on the occurrence of porphyrin bonded to SWCNT surface.It is known that not conjugated porphyrins having similar chemical structure to those used in this study are able to generate ROS when light irradiated and kill cellsvia US excitation . SWCNT–porphyrin behaves as a selective US-activated hybrid nanosystem. The thermal energy, coming from inertial cavitation, is efficiently transferred to the few porphyrin units linked to the nanotube surface, causing molecule excitation, then, similarly, to what happens in a photo-induced electron transfer (PET) process, the electron transfer between excited porphyrin (electron-donator) and carbon nanotube aromatic surface (electron-acceptor) stabilises the separated-charge state, increasing the probability of molecular oxygen to react with the excited system and facilitating the ROS production citation .24 A 40-fold lower dose of porphyrin grafted on SWCNT is sufficient to induce a similar cell toxicity. So far, various porphyrin nanocarriers have been investigated due to their inherent ability to improve the triggering effect US has on the sonosensitizing agent.36,37 However, CNT-based nanocarriers shows unique properties, such as the photoinduced electron transfer, making them an intriguing multifunctional hybrid nanoplatform suitable for the development of the next generation of US responsive nanosystems. Moreover, the results reported herein are supported by the work of Yumita et al.38,39 highlighting the great potential of CNT-based nanosystems in exploiting the US-mediated ROS generation, representing the pivotal step of sonodynamic applications.This feature can be further highlighted by comparing cell toxicity induced by our SWCNT–porphyrin system with that induced by-decorated polymeric core–shell nanoparticle (PCNP), under the same experimental conditions.i.e. pericyclic reaction), produces very small amount of porphyrin grafted on SWCNT and, especially, the three nanosystems show very similar biological behaviour, the structural and morphological properties of these nanohybrids were assessed only on SWCNT–1, using several complementary physico-chemical characterization techniques. The results were compared with those obtained with pristine carbon nanotubes (SWCNT). Atomic force microscopy (AFM) and transmission electron microscopy (TEM) images  and showed a minimum width of 7 nm. No significant morphological modifications were induced by the grafting procedure.Since the synthetic procedures share the same pressure and temperature conditions, same type of reaction mechanism  images  were use) images  are cons21 Raman spectroscopy confirmed that the functionalization process does not significantly modify SWCNT structure . The lowest frequency peak  is a first-order Raman-allowed feature originating from the in-plane stretching of the C–C bond. The highest frequency peak (G′ band) is usually assigned to a second order two-phonon feature of crystalline graphite and, generally, of all sp2-carbon systems. In SWCNT, the rolled structure of the tube breaks the two-dimensional graphene symmetry and up to six high-energy tangential modes are allowed for the G-band. Those modes can be practically treated considering only two intense superimposed peaks labelled G+, for atomic displacements along the tube axis, and G−, for modes with atomic displacement along the circumferential direction. The G+ frequency is constant with diameter for metallic and semiconductor tubes, while G− can be used to infer about the strain affecting CNT structure, including nanotube curvature. At lower Raman shift (ca. 1350 cm−1), a peak is observed in the presence of defects that discontinue the otherwise perfect sp2 graphite structure. This peak is conventionally named D-band, where D stands for defect or disorder. D-band in SWCNT is commonly used to discuss the disorder-induced features related to symmetry breaking of the sp2 configuration of graphene sheet. At Raman shift ranging from 160–280 cm−1 radial breathing mode (RBM) are usually observed for SWCNT. ca. 1580 cm−1 showing the two G+ and G− components at 1583 and 1516 cm−1, respectively, and the G′-band at 2645 cm−1. The sharpness and moderate downshift of the G− band of the pristine SWCNT signals the semiconducting nature of the tube used in this work. The D-band is observed at 1326 cm−1. A qualitative comparison of the peak shapes and frequencies between pristine and conjugated SWCNT evidenced that the structural features of the tube were largely preserved during the porphyrin conjugation. Because absolute intensity measurement is a difficult task in Raman spectroscopy, the normalized intensity ID/IG ratio was used to quantitatively describe the amount of disorder . All the conjugates obtained showed a negligible increase of the relative intensity of the D-band with respect to G+-band (ID/IG ratio), exhibiting ratios between 0.08 and 0.16. To comparative analyse this range of values, one should consider that a defective SWCNT might exhibit ID/IG ratio > 1.6.40 The relative intensity and the downshift of G−-band is virtually the same for all samples, indicating that conjugation-induced strain did not significantly affect tube curvature. Furthermore, the functionalization did not induce shifts in the main features of RBM at 186 and 270 cm−1, further confirming the stability of tube diameter over conjugation. Overall, the Raman spectra of the pristine and the variously characterized SWCNTs were superimposable and the structural integrity of tubes after functionalization further confirmed.As previously observed on reduced graphene oxide grafted with porphyrin,tructure . Raman sdisorder  eventualmeso-tetrakis(4-carboxyphenyl)porphyrin was purchased from Frontier Scientific, Pure HiPco single walled carbon nanotube was purchased from Unidym. MW assisted reactions were carried out in a monomodal reactor Monowave 300 (Anton Paar) equipped with a monitoring system of temperature, pressure and power input. The CombiFlash RfsTeledyne (ISCO) purification system was used to purify all crude reactions on silica gel. Analytical and preparative HPLC-MS was performed using a FractionLink autopurification system (Waters) equipped with a 2996 photo diode array detector and Micromass ZQ detector (ESCI hybrid ionization source). NMR spectra were recorded on an Avance 300 or on an Avance 600 spectrometers (Bruker) operating respectively at 7 T and 14 T, dissolving samples in proper deuterated solvent. Chemical shifts were referenced per residual solvent signals. MS spectra were carried out using electrospray ionization (ESI) or by atmospheric pressure chemical ionization (APCI), in positive ion mode, on a Micromass ZQ spectrometer (Waters).All reagents and solvents were purchased from Sigma-Aldrich and used without further purification, −1) by a Pyris 1 ultra-micro-balance; the gases evolved during the heating ramp were continuously monitored by a Spectrum 100 FTIR spectroscope (Perkin Elmer). The gas evolved during the heating ramp was piped (gas flow 65 ml min−1) via pressurized heated (280 °C) transfer line (Redshift S.r.l.) and analysed continuously by the FTIR , equipped with a thermostated (280 °C) conventional gas cell. The spectra were acquired in the 4000–600 cm−1 wavenumber range with a resolution of 0.4 cm−1 and analysed with the Spectrum software (Perkin Elmer). Temperature-resolved infrared profiles of the identified volatiles evolved from samples were obtained from the intensity of a representative peak of the investigated species.Thermogravimetric analysis: TGA analyses were carried out under dynamic nitrogen atmosphere  has been focused on a 1 μm × 1 μm wide portion of the SC. A polarized solid-state Nd laser operating at a wavelength of 532.11 nm and power of 80 mW and a CCD air-cooled detector operating at −70 °C were used. Calibration of the instruments was performed by measuring the Stokes and anti-Stokes bands of the Si band at 520.7 cm−1. Samples were placed on a polished stainless-steel slide and a 50× objective delivering a power of ca. 15 mW on the sample was used. Spectra were acquired with a spectral resolution of ca. 2 cm−1 and an integration time spanning from 100 to 400 seconds.TEM analyses: the morphology of the samples was investigated in the nanometric range by means of a JEOL 3010-UHR transmission electron microscope (TEM) equipped with a LaB3N4 Cr–Au back-coated cantilevers with typical resonance frequency 190 kHz, force constant 45 N m−1, tip radius lower than 35 nm and full tip cone angle 40°. Due to the softness of the samples, all measurements were performed in alternated contact mode.Atomic force microscopy (AFM): AFM height profile measurements were performed using a DME SPM Microscope (DME Igloo) equipped with a DS95-50E scanner (scan volume 50 × 50 × 5 μm). The data were acquired using MikroMasch Ultrasharp NSC16/Si−2 for 5 min, measured using Actinic UV-meter (Jelosil).Light exposure apparatus: a custom system for LB exposure with fixed source-tube positioning and light diffuser all-around cell culture was used. The light-emitting source of the system is based on InGaN light-emitting diodes (Cree Inc.) with 20 mW max radiant power at a central wavelength of 405 nm. The energy fluency rates of the light radiation were adjusted to 15 mW cmf0 = 1.866 MHz connected to a power amplifier  and a function generator . A mechanical adaptor was built to connect the 1 cm diameter polystyrene tube containing the cells suspended in PBS. When filled with ultrapure water, the adaptor creates highly reproducible measurement conditions at a fixed cell tube distance from the transducer.US exposure apparatus: the US field was generated by a plane wave transducer in continuous wave at Cell viability was evaluated by absorbance measurements (450 and 620 nm as reference wavelength) on a microplate reader .30 Whereas the procedure to obtain compound 2 is here reported.Compound 1 and 3 were synthesized accordingly with reported procedures.29 developed by authors, in 2% global yield.Compound (1) was synthesized accordingly to previously described procedure,1H NMR  δ 9.18 , 8.25 , 7.39 , 7.08 , −2.55 ; APCI-MS+: m/z calcd for C36H22N4O4, 574.16; found 575.48 [M + H+].4  was slowly added. The mixture was stirred at RT for 18 h, then water (155 ml) was added. The two phases were separated and the aqueous layer was washed with ethyl acetate (2 × 50 ml). The organic layers were then combined and washed with a 5% NaHCO3 solution (2× 100 ml), dried over sodium sulphate and solvent was removed to obtain (a) in 94% yield (11.25 g). 1H NMR  δ 7.32 , 7.19 , 4.99 , 3.82 , 3.00 , 1.72 , 1.50–1.15 ; 13C NMR  δ 169.6, 156.8, 137.8, 129.0, 128.4, 128.3, 83.5, 80.2, 65.8, 52.9, 30.6, 29.4, 28.1, 22.0; ESI-MS+: m/z calcd for C18H28N2O4, 336.42; found 337.19 [M + H+].Lys-CBz  was dissolved in terz-butyl acetate (170 ml) and HClO2Cl2 and heated under reflux until complete dissolution. The solution was allowed to reach ambient temperature and Na2CO3  was added. Mixture was stirred for 1 h then a solution of BOC2O  in CH2Cl2 (20 ml) was slowly added. The reaction proceeded under stirring overnight, then it was filtered ad washed with 10% NaHCO3 solution (3 × 20 ml) and with water (2 × 20 ml). Organic phase was dried over sodium sulphate; solvent was removed under reduced pressure and compound (b) was collected in 87.5% yield (1.13 g).Compound (a)  was added to 50 ml of CH1H NMR  δ 7.19 , 4.94 , 3.99 , 3.02 , 1.73–0.96 ; 13C NMR  δ 171.8, 156.5, 155.5, 146.7, 128.4, 128.0, 127.9, 85.0, 81.6, 79.5, 66.4, 53.7, 32.42, 31.1, 28.3, 27.9, 22.3; ESI-MS+: m/z calcd for C23H36N2O6, 436.26; found 459.20 [M + Na+].2 was removed under vacuum, NaOH (2 M) was added until pH = 9, methanol was evaporated under reduced pressure ad resulting aqueous phase was extracted with ethyl acetate (3× 50 ml). Organic layer was dried over sodium sulphate and solvent evaporated to obtain compound (c) in 15% yield. Acidimetric titration indicates 85% of purity.Compound (b)  was dissolved in methanol (100 ml) and Pd/C  was added. The mixture was stirred at room temperature under hydrogen atmosphere (atmospheric pressure) for 48 h. Then, it was filtered and solvent was removed under reduced pressure obtaining a white solid (2.4 g). Acidimetric titration indicates that this solid was formed by compound (c) (40%) and its carbamic acid form (60%). The solid was dissolved in methanol, cooled down to 5 °C in an ice bath, and aqueous HCl  was slowly added. The CO1H NMR  §§Some impurities were observed; however chemical shifts refer to most abundant compound.δ 3.82 , 2.48 , 1.82–0.70 ; ESI-MS+: m/z calcd for C15H30N2O4, 302.22; found 303.19 [M + H+].meso-tetrakys-(4-carboxyphenyl)-porphyrin  was dissolved in a mixture of CH2Cl2 (30 ml) and THF (10 ml), then EDC  and DMAP  were added. The mixture was stirred at RT for 30 min and a solution of compound (c)  in CH2Cl2 (30 ml) was slowly dropped. The reaction progress was monitored by TLC , HPLC  and ESI-MS. After 4 h the reaction was completed and the mixture was separated by flash chromatography  and the product containing fraction was further purified by preparative HPLC-MS  obtaining 50 mg of (2) in 34% yield. The 1H-NMR spectrum shows the presence of (2) in mixture with its partially deprotected derivatives which do not compromise its use in the next deprotection step. The solid was then dissolved in CH2Cl2 (20 ml) and TFA (3 ml) was slowly added. The mixture was stirred overnight and solvent and excess of TFA were evaporated under reduced pressure obtaining a blue/green solid .Commercial 1H NMR  δ 8.82 , 8.31 , 4.07 , 3.62 , 2.06 , 2.29–1.5 ; 13C NMR  δ 136.8, 131.4, 127.2, 53.5, 40.4, 30.8, 29.9, 23.2; ESI-MS+: m/z calcd for C72H78N12O12, 1302.59; found 1304.0 [M + H+], 652.6 [M + 2H+].30 developed by authors, in 46%. Yield (28.5 mg) 1H NMR  δ 8.50 , 8.28 , 7.96 , 7.36 , 7.31  6.33 , 3.66–3.54 , 3.07 .Compound (3) was synthesized accordingly to previously described procedure,2Cl2 (3× 4 ml). The black solid was recovered and dried at 100 °C for 48 h (21 mg). The final product was characterized by Raman spectroscopy, TGA, AFM and TEM.SWCNT (20 mg), compound 1  and DMF (4 ml) were placed in a specific glass tube (Anton Parr G10). The tube was placed in the MW oven, sealed and heated at 100 °C (temperature-controlled MW irradiation with max power of 600 W) for 2 h. The mixture was then cooled to RT, centrifuged and washed with DMF (3 ml) and CH2Cl2 (3× 4 ml). The solid was recovered and dried at 100 °C for 48 h. The final product was characterized by Raman spectroscopy and TGA.SWCNT (30 mg), octanal , compound 2  and DMF (3 ml) were placed in a specific glass tube (Anton Parr G10). The tube was placed in the MW oven, sealed and heated at 120 °C (temperature-controlled MW irradiation with max power of 600 W) for 2 h. The mixture was then cooled to RT, centrifuged and washed with DMF (3 ml) and CH2Cl2 (3× 4 ml). The black solid was recovered and dried at 100 °C for 48 h (14 mg). The final product was characterized by Raman spectroscopy and TGA.SWCNT (10 mg), compound 3  and DMF (3 ml) were placed in a specific glass tube (Anton Parr G10). The tube was placed in the MW oven, sealed and heated at 100 °C (temperature-controlled MW irradiation with max power of 600 W) for 2 h. The mixture was then cooled to RT, centrifuged and washed with DMF (3 ml) and CH−1 in PBS pH 7.4) and bath sonicated for 3 h. Finally, unbound conjugate was removed by repeat filtering and washing with PBS .SWCNT (1 mg) were suspended in 2 ml of mPEG-DSPE solution  1640 and RPMI 1640 + 10% FBS ] and bath sonicated for 1 h then stability within 30 days was evaluated. The suspension appears stable after so long period  heat-inactivated fetal calf serum (Sigma-Aldrich) in a humidified atmosphere of 5% CO2 air at 37 °C.Human colorectal cancer cell line, HT-29 , was cultured in RPMI-1640 medium, supplemented with 2 mM −1 of SWCNT and porphyrin-loaded SWCNT  in polystyrene tubes for light beam (LB) or US exposure.Cells in the exponential growth phase were harvested with 0.25% trypsin and normalized to 5.0 × 105 cells in 3 ml of medium containing 10% of percoll® and 25 μg ml−2, for 5 min. The light intensity was measured using Actinic UV-meter .LB exposure was carried on by custom system, adjusting energy fluency rates of light radiation at 15 mW cmf0 = 1.866 MHz and with an energy density corresponding to 0.008 mJ cm−2; energy supplied to the cells did not increase the medium temperature (maximum temperature recorded was 33 °C).US cells irradiation was carried out at 3 HT-29 cells were seeded in 100 μl of growth medium in replicates (n = 8) in 96-well culture plates . At 24 and 48 h, WST-1 reagent (10 μl) was added and the plates were incubated at 37 °C in 5% CO2 for 1.5 h. Well absorbance was measured at 450 and 620 nm (reference wavelength) on a microplate reader Asys UV 340.The effect that porphyrin loaded SWCNT had on HT-29 cell proliferation was evaluated by WST-1 cell proliferation assay . After various treatments, 2.0 × 10p < 0.05.Data are shown as average values ± standard deviation of three independent experiments. Statistical analyses were performed on Graph-Pad Prism 6.0 software ; two-way analysis of variance and Bonferroni's test were used to calculate the threshold of significance. Statistical significance was set at −1, respectively. The suspension was introduced in a polystyrene tube  and exposed to US (1.866 MHz) under the same experimental conditions employed in cell experiments (namely 0.008 mJ cm−2 for 5 min). Sonication was carried out using also different sonotrode frequency (22 kHz): 2 ml of suspension were introduced in polystyrene tubes and exposed to ultrasound (10 kJ cm−2 for 1 min) employing a sonicator Sonopuls HD3100 equipped with a 73 MS probe . After the US treatment, the suspension was filtered on a cellulose acetate membrane  and TA-OH fluorescence  was measured on the clear solution by a FLx800 fluorescence reader . The results were compared to a blank solution without US irradiation and on US-irradiated pristine SWCNT (25 μg ml−1) under the same experimental conditions. Three replicates for each experiment were carried out and the results were expressed as the mean values ± SD of the separate determinations and analysed by a one-way Analysis of Variance (ANOVA) and Tukey's test. p < 0.05 was considered significant.Terephthalate (TA) solution (8 mM) was prepared dissolving disodium terephthalate in Milli-Q ultrapure water. A stable SWCNT-1 suspension was obtained suspending SWCNT–1 in DMSO (purity 99.9%). TA solution was added to the DMSO : water (1 : 3) suspension of SWCNT–1. The concentration of TA and SWCNT–1 in the final mixture was 4.0 mM, and 25 μg mlvia Diels–Alder cycloaddition (compounds SWCNT–1 and SWCNT–3) and azomethine ylide 1,3-dipolar cycloaddition (SWCNT–2) were thoroughly characterized comparing pristine SWCNT.To assess the structural and morphological modifications possibly induced by conjugation on SWCNT, several physico-chemical characterization techniques were complementary used. Raman spectroscopy , thermogper se in vitro but kills up to 75% HT-29 colon cancer cell under low-power US irradiation. The selectivity (no effect under LB) and high efficiency (40-fold higher than PCNP) of these hybrid nanosystems are here demonstrated for the first time and it might be due to the increase of the separated-charge state lifetime, which promotes ROS production and, in turn, may impair cell proliferation. The lack of photo-induced effects of these SWCNT–porphyrin could allow to overcome the big limit of skin photosensitivity. The here described hybrid nanosystem represents an interesting model for a further development of selective US-activated anticancer sonosensitizer.In this study, we propose a novel covalently grafted SWCNT–porphyrin hybrid nanosystem, which is biologically active only upon US irradiation thanks to the synergistic action of the two bonded entities. The hybrid nanosystem is non-toxic There are no conflicts to declare.RA-010-D0RA03944F-s001