Under near-infrared laser irradiation, CVS NPs not only generate hyperthermia to ablate tumor cells but also catalytically yield superoxide radicals and induce endogenous NADH oxidation through the Seebeck effect. Notably, CVS NPs can accumulate inside mitochondria and deplete NADH, reducing ATP synthesis by competitively inhibiting the function of complex I, thereby down-regulating the expression of heat shock proteins to relieve tumor thermotolerance. Both in vitro and in vivo results show notable tumor suppression efficacy, indicating that the concept of integrating PTET and mitochondrial metabolism modulation is highly feasible and offers a translational promise for realizing precise and efficient cancer treatment.Thermoelectric therapy has emerged as a promising treatment strategy for oncology, but it is still limited by the low thermoelectric catalytic efficiency at human body temperature and the inevitable tumor thermotolerance. We present a photothermoelectric therapy (PTET) strategy based on triphenylphosphine-functionalized Cu 3VS4 nanoparticles with copper ionic mobility are developed for photothermoelectric tumor therapy.Triphenylphosphine-modified Cu Together, the p-type ternary compound Cu3VS4 not only efficiently converts NIR light into heat but also has favorable thermoelectric performance at room temperature, showing great potential as a novel PTE material for PTET. Furthermore, Cu3VS4 NPs were modified by using distearoyl phosphoethanolamine–(polyethylene glycol)–triphenylphosphonium bromide (DSPE-PEG-TPP) to form Cu3VS4-TPP NPs (CVS NPs) with the mitochondrial-targeting ability for photoacoustic (PA) imaging–guided synergistic PTET/chemodynamic therapy (CDT) (−) play a role in the reduction of oxygen (O2) molecules to produce superoxide radicals (·O2−), while holes (h+) are involved in NADH oxidation. CVS NPs competitively inhibit the activity of mitochondrial respiratory complex I by depleting NADH, thereby suppressing the ATP supply and down-regulating the expression of HSPs. Last, the undesired heat tolerance of tumor cells is alleviated during PTET. This “domino effect” facilitates the cell to approach apoptosis. Moreover, in the mild acidic tumor microenvironment (TME), CVS NPs are capable of catalyzing hydrogen peroxide (H2O2) to yield toxic hydroxyl radicals (·OH) for CDT through a Fenton-like reaction. In both in vitro and in vivo measurements, we control the temperature gradient (15° to 55°C) by an external cold source and 808-nm laser excitation to maximize the temperature gradient, thus greatly potentiating the thermoelectric effect. These prominent PTE/chemodynamic features endow the CVS NPs with effective antitumor performance and negligible biosafety concerns, demonstrating the proof-of-concept application of “phonon-liquid” PTE materials for cancer therapy.In this work, the ternary copper-based chalcogenide compound Cupy (CDT) . Under 83VS4 NPs were prepared according to a modified method, in which the sulfur precursor solution was mixed with the metal precursor solution via a thermal injection strategy  pattern of Cu3VS4 NPs is presented in 3VS4 phase (PDF 00-011-0104) with the lattice parameter of a = 5.393 Å (3VS4 NPs were obtained through the utilization of x-ray photoelectron spectroscopy (XPS). The as-synthesized Cu3VS4 NPs mainly consisted of Cu, V, and S elements  and V5+ (45.1%) in Cu3VS4 NPs. Meanwhile, two peaks can be resolved in the Cu 2p XPS spectrum at the binding energies of 952.6 and 932.8 eV with the spin-orbit splitting of 19.8 eV, which were attributed to Cu 2p1/2 and Cu 2p3/2 states, severally . The results of thermogravimetric analysis (TGA) suggest that the proportion of DSPE-PEG-TPP in CVS is approximately 31.63% (fig. S2B). CVS NPs display a similar morphology as Cu3VS4 NPs with good dispersion and no obvious agglomerate (fig. S3A). Furthermore, as confirmed by dynamic light scattering (fig. S3B), the hydrodynamic size of CVS NPs in phosphate-buffered saline (PBS) is approximately 20.31 nm [polydispersity index (PDI) = 0.09 ± 0.06] with a positive charge of ~7.27 mV (fig. S4). In addition, the average size and the PDI of CVS NPs in PBS and RPMI 1640 + 10% fetal bovine serum over 7 days were evaluated (fig. S5). The CVS NPs exhibit a uniform hydrated particle size and demonstrate no obvious PDI variation in various solutions, confirming the excellent stability of CVS NPs in physiological conditions.The high-resolution V 2p XPS spectrum of Cu 7.45 eV . This reeverally . The pro−1 cm−1 (−1) increased considerably by 49.7°C upon 5 min of laser excitation (1.5 W cm−2). In contrast, there was only a slight increase in the temperature of pure water, indicating the high efficiency of CVS NPs in transforming NIR laser into thermal energy. Furthermore, CVS NPs have a definite dose-dependent behavior that facilitates the regulation of the PTET temperature gradient by adjusting the injected dose (ID). Moreover, a positive correlation was observed between the temperature of the CVS aqueous solution and the power density of laser, with the former increasing steadily as the latter was elevated –visible (vis)–NIR absorbance spectrum , in whic−1 cm−1 . Subsequ−1 cm−1 . It is nelevated . The staelevated . Besideselevated . Given tz direction. Considerable electric field enhancement was observed on two sides of the Cu3VS4 nanostructure , where the local and incident electric fields are denoted by Elocal and Ein, respectively. Moreover, the steady-state temperature distribution of the Cu3VS4 nanostructure in water (refractive index = 1.33) under 808-nm laser irradiation was simulated. The Cu3VS4 nanostructure exhibits a certain enhanced photothermal effect from the combination of heat generated by the sulfide itself and the interaction with z-directional electromagnetic waves, with the temperature difference appearing at two ends of the NPs (fig. S8). It is inferred that the high photothermal conversion efficiency of CVS NPs is due to the narrow bandgap and the intermediate band formed by the introduction of V ions. Owing to the transient optical response leads to the ultrafast nonradiative relaxation of carriers excited from the VB to the IB and the subsequent ultrafast thermalization of the NP lattice, which is consistent with previously reported photothermal models of other IB semiconductors  describes the ability of thermoelectric materials to convert a temperature difference into electrical voltage, defined as S = −ΔV/ΔT. To comprehensively gauge the thermoelectric conversion efficiency of thermoelectric materials, the dimensionless figure of merit (ZT) is defined asS represents the Seebeck coefficient, σ denotes the electrical conductivity, T is the absolute temperature, and κ represents the thermal conductivity (S2σ) or reducing thermal conductivity to establish a large temperature difference.In the thermoelectric conversion progress, the Seebeck coefficient (3VS4 NPs after spark plasma sintering (SPS) was measured. The electrical transport properties in the temperature region from room temperature (298 K) to 100°C (373 K) are shown in fig. S9. Cu3VS4 NPs exhibit excellent conductivity with the resistivity ρ as low as 1.58 milliohm·cm at room temperature. The conductivity σ can be calculated by taking the inverse of the resistivity (σ = 1/ρ), resulting in a conductivity of 63.4 × 103 S m−1 for Cu3VS4. It is obvious that the ρ increases smoothly from 300 to 358 K, after which it begins to increase rapidly, with ρ reaching 1.73 milliohm·cm, and σ is calculated to be 57.7 × 103 S m−1 at 373 K. The σ of Cu3VS4 decreases monotonically with increasing temperature, exhibiting a highly degenerate semiconducting transport behavior. The positive value of the S indicates the p-type semiconducting behavior of Cu3VS4, and as the temperature increases from 298 to 373 K, the S increases from 20.7 to 31.9 μV K−1 (fig. S9C). On the basis of the measured σ and S, the calculated PF at room temperature and 373 K are 27.1 and 58.5 μW m−1 K−1, respectively (fig. S9D). In addition, the thermal transport properties of Cu3VS4 are presented in fig. S10 (A to C), including specific heat capacity (CP), thermal diffusivity (D), and thermal conductivity (κ). The κ was examined at 298, 313, 335, 357, and 373 K, and the data are shown in fig. S10C. It can be observed that the κ value increases as the temperature rises, all of which are below 0.4 W m−1 K−1. The κ of Cu3VS4 is as low as 0.3 W m−1 K−1 at room temperature, which is extremely low for thermoelectric materials in the relatively low temperature range (fig. S11). The final calculated ZT values are presented in fig. S10D. Because of the moderate S and ultralow κ, the ZT value at room temperature is calculated to be 0.027, reaching 0.060 at 373 K.Initially, the thermoelectric performance of Cu3VS4, we further explore its intrinsic structure. It is well known that Cu3VS4 is an IB semiconductor, whose optical properties are dominated by the collective optical transitions between energy bands. The UV-vis diffuse reflectance spectrum was used to determine the optical properties of the synthesized CVS NPs. There is a broad continuous extinction starting from the NIR region, which extends throughout the visible range  and threefold degeneracy of the VBs  can be observed at the Γ point. Another threefold degeneracy happens at the top of the VB (R point), where they overlap near the Γ and R points because of the inclusion of both heavy bands and light bands. This band overlap, particularly at the bottom of the conduction band (CB)/IB and the top of the VB, is the main physical source for excellent thermoelectric performance , face center (labeled as A2), and body center (labeled as A3) sites. Path 1 represents the Cu ions at A1 sites that migrate directly to A3 sites; path 2 represents the transition path from A1 to the A2 sites, and path 3 refers to the movement of Cu ions from A2 to A3 sites , (101), and (111) crystal planes were simulated and shown in A3 sites . The dif·O2− generated by PTET was performed by nitro blue tetrazolium (NBT) assay, as NBT molecules could capture ·O2− and generate blue-violet NBT-formazan with a characteristic peak at ~560 nm (fig. S13). The characteristic absorption peak is gradually enhanced as the number of cycles increases  trapping in methanol , and the CB minima position is calculated on the basis of the equation ECB = EVB − Eg, where Eg, ECB, and EVB reveal the bandgap energy, CB potential, and VB potential, respectively. Considering the bandgap of Cu3VS4 as 1.20 eV, the CB potential is calculated to be −0.48 eV. As shown in fig. S14B, the obtained CB potential is more negative than the redox potential of O2/·O2− [−0.33 eV versus normal hydrogen electrode (NHE)]. The more negative CB potential suggests that the formation of ·O2− radical is possible.We next irradiated the CVS NPs with an 808-nm laser for 2 min (laser on), followed by natural cooling to room temperature (laser off). Five laser on/off cycles were executed to research the mechanism of PTET. The detection of ncreases . The elemethanol , further+, as vital cofactors of intracellular redox metabolism, participate in the OXPHOS pathway. The PTE oxidation of NADH was monitored via the UV-vis absorption spectrum. The characteristic absorption peak of NADH (~340 nm) decreases obviously with the increase of the NAD+ absorption peak  could lead to a remarkable temperature gradient, which may induce a thermoelectric signal. Therefore, to maximize the thermoelectric effect, in addition to the superior photothermal conversion capability of the material, an additional cooling source is required for a sharp temperature drop to ensure a high dT/dt rate  is built up through the photothermal conversion progress. The holes (h+) at the hot side of the p-type conductor migrate to the cold side because of their increased kinetic energy, and when enough holes are gathered at the cold side, a built-in electric field internal is formed in CVS. With the accumulation of holes, the built-in electric field is gradually enhanced and further prevents the diffusion of holes from the hot side to the cold side. When the carriers inside the NPs reach equilibrium, an electric potential difference (ΔV) is formed between the hot and cold sides of the CVS NPs. The excited electron-hole pairs at the temperature gradient could be effectively separated and reached the surface of CVS NPs to further participate in redox reactions, where electrons can be transferred to molecular oxygen generating ·O2−, and NADH is oxidized to NAD+ by holes.As all the above results, CVS NPs could generate ·OH from H2O2 decomposition was first examined using the o-phenylenediamine (OPD) assay, according to the fact that OPD can be oxidized by the highly reactive ·OH to give an orange color with the characteristic peak of oxidized-OPD (oxOPD) at ∼420 nm. Neither H2O2 nor CVS alone had a detectable effect on the absorbance increase of OPD (·OH generator. Under mildly acidic condition (pH = 5.5), CVS NPs induce a remarkable enhancement in oxOPD characteristic absorption, whereas only a low absorption peak is displayed under neutral conditions (pH = 7.4). Furthermore, ESR spectroscopy experiments affirm the higher production of ·OH under mildly acidic conditions, as evidenced by the distinct quadruple resonance peaks with a signal intensity ratio of 1:2:2:1 observed in the group (1) using DMPO as the spin trap , CVS NPs could catalyze the oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB) with the existence of H2O2 to generate blue oxidized TMB (oxTMB). The absorbance value of oxTMB at ~652 nm increased observably with the increasing H2O2 concentration . The acid-mediated generation of ·OH by CVS NPs could be ascribed to the oxidation of the lower oxidation state V4+/Cu+ to V5+/Cu2+ by H2O2 in the presence of H+. These results indicate that CVS NPs could act as a fascinating Fenton-like agent to produce ·OH for efficient CDT in an acidic environment. Together with the above excellent chemodynamic performance, the CVS NPs are expected to be used for ROS-based synergistic PTE/CDT.Because of the variable-valence V and Cu ions, we wondered whether CVS NPs would have a Fenton-like activity for CDT. The generation of e of OPD . Becausepin trap , a charantration , thus va3VS4 NPs to form Cu3VS4-PEG NPs and CVS NPs, respectively, in an attempt to explore whether TPP modification could enhance lysosomal escape and induce mitochondrial accumulation  as an ·O2− probe. Notably, the CVS + NIR + ice group displays the highest red fluorescence intensity compared to other groups, confirming the efficient ·O2− production . In addition, an ice pack was used as the cold source to control the temperature to quickly drop to 15°C, thus inducing a sharp temperature drop to ensure a high dc effect . After cfect  can be oxidized to paramagnetic Cu(II)  by H2O2 on the surface of CVS NPs, there is no doubt that CVS NPs can serve as a suitable agent for TME-responsive T1-weighted magnetic resonance (MR) imaging. A concentration-dependent signal enhancement effect with the longitudinal relaxation (r1) value of 4.07 mM−1 s−1 is observed after incubation with H2O2, higher than that of CVS NPs without H2O2 addition . Thereafter, time-dependent MR imaging capability was evaluated in 4T1 tumor–bearing mice after intravenous injection of CVS NPs at different time intervals . The T1-MR signals of the tumor are markedly enhanced with the injection time extension, whose intensity reaches a maximum level after 12 hours of intravenous injection (−2) irradiation, the temperature of the tumor can be raised to 54.8°C within 5 min  = 0.24 hours and t1/2(β) = 3.75 hours. The moderate circulation time is beneficial to the accumulation of NPs in the tumor. High levels of Cu could be detected in feces of mice after intravenous injection of CVS NPs (fig. S22B), indicating that CVS NPs are easily excreted through fecal and renal pathways, thus avoiding the long-term toxicity of the NPs in mice.Considering that the diamagnetic Cu(I)  on the right back of each mouse (n = 10 per group) and treated accordingly: (i) control (PBS), (ii) NIR, (iii) CVS NPs, (iv) CVS NPs + NIR, and (v) CVS NPs + NIR + ice pack (five cycles). For mice in groups (iii) to (v), CVS NPs  were firstly injected intravenously, where group (iii) was not further treated, group (iv) was subjected to an 808-nm laser (1.0 W cm−2), while group (v) was successively irradiated with a 808-nm laser and cooled by an ice pack for five cycles at 12, 24, and 48 hours after injection. Notably, an ice pack was used as the cold source to control the temperature to quickly drop to 15°C, thus inducing a sharp temperature drop to ensure a high dT/dt rate. The real-time temperature at the tumor region in the whole heating/cooling process was observed using a thermal imaging camera. Next, the tumor growth profiles were monitored throughout the entire treatment, as illustrated in To further investigate the in vivo antitumor efficacy of CVS NPs, a 4T1 tumor–bearing female BALB/c mice xenograft model was established by subcutaneously injecting 4T1 cells  CVS NPs could catalytically generate ·O2− and induce the oxidation of endogenous NADH through PTE effect, which decrease the secretion of ATP during treatment to further down-regulate the expression of HSP70. Furthermore, the body weights of mice in each group were recorded every 2 days throughout the 14-day assessment period. There was no notable difference in body weights of mice in each group, which displays that the injection of CVS NPs has no apparent systemic toxicity (·O2− in the tumor after different treatments (fig. S23). Contrasted with the weak fluorescence in tumor slices from control, NIR, and CVS groups, the CVS + NIR group shows an obvious fluorescence signal, and the strongest fluorescence is observed in the CVS + NIR + ice group, suggesting the efficient production of ROS and ·O2−. Subsequently, a commercial NADH/NAD+ assay kit was used to evaluate NADH/NAD+ redox ratios in tumor regions of mice (fig. S24), and the expression level of HSP70 in different groups was examined using an immunofluorescence assay  The CVS NPs could convert endogenous Htoxicity . The mictoxicity . To furtce assay . In the  CVS NPs . In acco CVS NPs . Through3VS4 nanostructure were calculated by DFT calculations: a threefold degeneracy at the top of the VB and a relatively low diffusion barrier of Cu ions within the lattice, resulting in an ultralow thermal conductivity of 0.3 W m−1 K−1 at room temperature. The obtained CVS NPs exhibit excellent photothermal conversion efficiency (32.78%) and ideal PA imaging capability exposed to an 808-nm laser. Meanwhile, the electrons and holes excited by the built-in thermoelectric field can effectively engage in redox reactions on the surface of CVS NPs, generating ·O2− and oxidizing NADH into NAD+. Notably, the depletion of endogenous NADH through the PTE effect triggers a domino effect: The function of mitochondrial respiratory complex I can be competitively inhibited and substantially leads to impairment of OXPHOS, cutting off the ATP supply and eventually causing the down-regulation of HSP70 levels. In addition, in the specific acidic microenvironment of the tumor, CVS NPs produce highly toxic ·OH for CDT through a Fenton-like reaction with H2O2. In vivo experiments guided by the designed treatment protocols show that CVS NPs synergistically induced apoptosis, efficiently suppressing tumor growth with a inhibition rate of 98.28% in the CVS plus NIR and ice pack group. Together, this work not only provides a simple but efficient strategy to conquer the limitations of traditional PTT through ROS-based synergistic PTET/CDT but also validates the great potential of mitochondrial metabolism modulation in the field of cancer therapy.In this proof-of-concept study, we introduced phonon-liquid CVS NPs, which have a prominent PTE effect to overcome the restrictions of conventional thermoelectric therapy, including low thermoelectric catalytic efficiency at human body temperature and inevitable tumor thermotolerance. The thermal and electrical transport properties of Cu3 , trioctylphosphine (TOP) , and 1-octadecene (ODE) (7 ml) were introduced into another 50-ml three-neck flask and dried under vacuum at 110°C for 30 min through a Schlenk line. The temperature was increased to 280°C with a heating rate of 10°C min−1 under a protective atmosphere of nitrogen. Afterward, the sulfur precursor solution (3.4 ml) was rapidly injected into the metal precursor solution. After injection, the temperature was allowed to recover, and the final solution was maintained at the target temperature for 20 min, after which it was cooled down to room temperature. The resulting NPs were washed three times with a mixture of ethanol and hexane in a volume ratio of 1:3 to remove excess ligands and unreacted precursors. The resulting precipitate was dissolved in hexane and stored in a glove box for further studies.The synthesis started with the preparation of sulfur precursor. 1-Dithiothreitol (DDT) (12 ml) and oleylamine (OLAM) (5 ml) were mixed in a 50-ml three-neck flask, then dried under vacuum at 110°C for 30 min and heated to 160°C under nitrogen for 30 min, and then cooled naturally. Meanwhile, metal precursors were prepared. Specifically, CuI , V(acac)3VS4 NPs in hexane were collected by centrifugation at 13,000 rpm for 10 min. Cu3VS4 NPs (20 mg) and DSPE-PEG-TPP (20 mg) were mixed in dichloromethane (10 ml) under magnetic stirring for 12 hours; then, the solvent of dichloromethane was removed by a rotary evaporator. Then, the as-prepared Cu3VS4-PEG-TPP NPs (CVS NPs) were obtained after centrifuging three times and washed with deionized water. For the synthesis of FITC-labeled CVS NPs, Cu3VS4 (20 mg), DSPE-PEG-TPP (20 mg), and DSPE-PEG-FITC (1 mg) were mixed in dichloromethane (10 ml) and stirred in the dark for 12 hours. For FITC-labeled Cu3VS4-PEG NPs synthesis, Cu3VS4 (20 mg), DSPE-PEG (20 mg), and DSPE-PEG-FITC (1 mg) were mixed in dichloromethane (10 ml) and stirred in the dark for 12 hours. Other protocols were the same as those described above. The resulting NPs were redispersed in PBS and kept in the dark before use.The Cu−5 eV and −0.01 eV/Å.DFT calculations were carried out using the VASP package (2SO4 aqueous solution (0.5 M) using a Pt plate as the counter electrode and Ag/AgCl as a reference electrode. The working electrode was fabricated by depositing the suspension (20 μl) prepared from CVS NPs (10 mg) mixed with Nafion  in ethanol onto a fluorine-doped tin oxide (FTO) glass.The thermoelectric current of CVS NPs under temperature variations was measured by an electrochemical analyzer (CHI660E) in the Na−1) and NADH solution (50 μg ml−1) with an 808-nm NIR laser (1.0 W cm−2) for 2 min (laser on), followed by natural cooling to room temperature (laser off) for a total of five laser on/off cycles. The absorbance changes of NADH at 340 nm and NAD+ at 260 nm were recorded using a UV-vis spectrophotometer. Subsequently, heating/cooling thermal cycles were performed for 3 ml of the mixed solution of CVS NPs (100 μg ml−1) and NADH (50 μg ml−1), in which the temperature variations were manipulated alternately by a hot-water bath (55°C) and a cold-water bath (15°C) apparatus. The CVS solution was wrapped in tin paper to avoid interference from external light.To explore the oxidation of NADH during PTET, we irradiated a mixture of CVS NPs solution  in dimethyl sulfoxide was mixed with CVS NPs  in PBS. Similar to the above-mentioned procedure, five laser on/off cycles were performed, and the absorption at 680 nm was measured on a UV-vis spectrophotometer.We verified the generation of ·OH by CVS NPs through CDT. Typically, OPD (1.0 mM) and CVS NPs (20 μg ml−1) were incubated in PBS solution containing 0.5 mM H2O2 with pH set to 5.5 or 7.4. For comparison, OPD mixed with CVS NPs or H2O2 were also detected. The absorbance changes of OPD at 420 nm were recorded. In addition, CVS NPs (20 μg ml−1) were treated with various concentrations of H2O2  in 2 ml of PBS solution (pH 5.5) containing TMB (0.5 mM). After coincubation for 15 min, the absorbance of TMB at 652 nm was monitored to detect the generation of ·OH.OPD and TMB were used to detect the production of ·O2− and ·OH, respectively. For the detection of ·O2−, CVS NPs (20 μg) were mixed with DMPO  in methanol (1 ml) for five laser on/off cycles or three heating/cooling thermal cycles, similar to above mentioned procedure. To detect ·OH, CVS NPs (20 μg) and DMPO  were mixed in PBS (1 ml) with H2O2 (0.5 mM), pH = 5.5 or 7.4. For comparison, DMPO mixed with CVS NPs or H2O2 were also detected.Quantitative analysis of ROS production was performed on an ESR spectrometer setup with a center field of 3450 G using 1.0-mm quartz tubes. DMPO dispersed in methanol and water were used to detect 5 cells each well and cultured overnight for adherence. Then, cells were incubated with a medium (1 ml) containing FITC-labeled Cu3VS4-PEG NPs (100 μg ml−1) or CVS NPs (100 μg ml−1) for 1 hour and 4 hours. Subsequently, the cells were stained with commercial LysoTracker Red DND-99 (100 nM) or Mito-Tracker Red FM (100 nM) according to the manufacturer’s guidelines, and then counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 15 min. After incubation, the 4T1 cells were rinsed with PBS several times and fixed with glutaraldehyde (2.5%). Last, the fluorescence images were recorded using a CLSM, and the colocalization analysis was conducted using the ImageJ software.The subcellular localization of the NPs was investigated by confocal microscopy. Briefly, 4T1 cells were seeded in a six-well plate containing 28-mm cover glass at 1 × 10−1) for 24 hours. MTT assay was used to test the cytotoxicity following a standard protocol. The therapeutic effect of CVS NPs was further studied. Briefly, 4T1 cells were treated with (i) control, (ii) NIR, (iii) ice, (iv) CVS, (v) CVS + NIR, and (vi) CVS + NIR + ice. The content of CVS in groups (iv), (v), and (vi) was 100 μg ml−1. Groups (ii) and (v) were irradiated with an 808-nm laser (1.0 W cm−2) for 10 min. Groups (iii) and (vi) used an ice pack as a cold source in a water bath to control the temperature to drop to 15°C quickly. While group (iv) was under 808-nm laser (1.0 W cm−2) irradiation for 2 min and cooled by an ice bath for five cycles. Subsequent treatments were conducted with the same experimental conditions as mentioned above.Cytotoxicity evaluation of CVS NPs was performed on 4T1 mammary cells via the standard MTT assay. 4T1 cells were treated with different concentrations of CVS NPs  for 10 min, while group (iv) was under irradiation for 2 min and cooled by an ice bath for five cycles.4T1 cells were seeded into six-well dishes and cultured overnight. Then, the cells were treated with different formulations: (i) control, (ii) NIR, (iii) ice, (iv) CVS, (v) CVS + NIR, and (vi) CVS + NIR + ice. The content of CVS in groups (iv), (v), and (vi) was equivalent. Groups (iii) and (vi) used ice packs as a cold source in a water bath to control the temperature to quickly drop to 15°C. After 4 hours of incubation, group (v) was irradiated by 808-nm laser irradiation  for 20 min. The cell fluorescence images were performed by a CLSM (Leica TCS SP8) with 488-nm laser excitation.·O2− evaluation, after 4T1 cells accepted the above treatments, the culture medium was replaced with DHE staining solution (30 μM) and cultured for another 40 min, followed by the nucleus being counterstained by DAPI for 15 min. Then, the cells were imaged using CLSM.For intracellular For mitochondrial integrity assay, after the treatments above, 4T1 cells were incubated with JC-1 staining solution  following the manufacturer’s protocol. The cells were then photographed using fluorescence microscopy in the red channel for J-aggregates and green channel for J-monomer, separately.For immunofluorescence staining, 4T1 cells were treated with (i) control, (ii) NIR, (iii) 55°C incubation, (iv) CVS, (v) CVS + NIR, and (vi) CVS + NIR + ice, followed by fixation in 4% paraformaldehyde for 15 min, and then permeabilized with 0.3% Triton X-100 for 30 min. Nonspecific binding was blocked using 5% bovine serum albumin. The cells were then incubated with primary antibodies of anti-HSP70 for 2 hours, while the cytoskeleton was labeled using CoraLite488-conjugated β-actin monoclonal antibody. After washing three times with PBS, the corresponding Alexa Fluor 555–conjugated anti-rabbit immunoglobulin G secondary antibody was added and incubated for 1 hour. Last, the cell nucleus was stained with DAPI and examined using CLSM.For live/dead cell staining, after 4T1 cells accepted the above treatments, the treated cells were stained with Calcein AM (2 μM) and PI (4 μM) for 15 min. After fixing the cells with glutaraldehyde and washing them with PBS, all fluorescence images were acquired via CLSM.−1) for another 12 hours, respectively. In particular, an 808-nm laser (1.0 W cm−2) was used for the CVS + NIR group for 10 min during the incubation. In CVS + NIR + ice group, ice was used as a cold source in a water bath to control the temperature to drop to 15°C quickly, and cells were irradiated by an 808-nm laser (1.0 W cm−2) for 2 min and cooled by an ice bath for five cycles. The NADH content of each group was obtained using the NADH/NAD+ Assay Kit (Beyotime Institute of Biotechnology) according to the manufacturer’s instructions.4T1 cells were seeded in six-well plates and incubated for 24 hours. Then, the cells were further incubated with various concentrations of CVS NPs solution  and cultured overnight. Fresh medium (1 ml) containing CVS NPs with various concentrations  was added to each well and incubated for 12 hours. Then, an 808-nm laser (1.0 W cm−2) was used for the CVS + NIR group for 10 min during the incubation. In the CVS + NIR + ice group, ice was used as a cold source in a water bath to control the temperature to quickly drop to 15°C, and cells were exposed to an 808-nm laser (1.0 W cm−2) for 2 min and cooled by an ice bath for five cycles. Then, the ATP level in each group was assessed using the ATP assay kit. Briefly, the cells were collected and lysed in RIPA buffer and centrifuged at 12,000 rpm for 10 min. Then, a supernatant (20 μl) was mixed with ATP detection working solution (100 μl) in opaque-walled 96-well plates, and luminescence was monitored at 560 nm using a luminometer.4T1 cells were seeded each well on a six-well plate  control, (ii) NIR, (iii) ice, (iv) CVS, (v) CVS + NIR, and (vi) CVS + NIR + ice. The content of CVS in groups (iv), (v), and (vi) was 100 μg ml−1. Groups (ii) and (v) were irradiated with an 808-nm laser (1.0 W cm−2) for 10 min. Groups (iii) and (vi) used ice packs as a cold source in a water bath to control the temperature to drop to 15°C. While group (iv) was irradiated by the 808-nm laser (1.0 W cm−2) for 2 min and cooled by an ice bath for five cycles. All treated cells were trypsinized, washed, and quantified by an annexin V–FITC/PI apoptosis detection kit by a flow cytometer (BD Accuri C6).4T1 cells were seeded in a six-well plate at an initial density of 1 × 105 cells per well). For HSP assays, the experiment was divided into six groups: (i) control, (ii) NIR, (iii) 55°C incubation, (iv) CVS, (v) CVS + NIR, and (vi) CVS + NIR + ice. For Bax, Bcl-2, pro–caspase-3, and cleaved–caspase-3 assays, the experiment was divided into five groups: (i) Control, (ii) NIR, (iii) CVS, (iv) CVS + NIR, and (v) CVS + NIR + ice. The 4T1 cells were collected using trypsin and lysed in a lysis buffer. Then, the proteins were separated by SDS–polyacrylamide gel electrophoresis gel electrophoresis and then transferred into a polyvinylidene fluoride membrane. After blocking with 5% dried skimmed milk for 1 hour, the samples were stained with the corresponding primary antibody  overnight and anti–β-actin antibody as the loading control, followed by cultivation with the goat anti-rabbit Dylight-800 antibody for 1 hour. Afterward, the membrane was visualized by an Odyssey CLx Image Studio.4T1 cells were preseeded in a six-well culture plate (1 × 106) dispersed in PBS (100 μl) on the right back of each BALB/c mouse and used for the following experiments when the tumor volume approached 60 mm3.Female BALB/c mice (4 weeks old) were obtained from Beijing Vital River Laboratory Animal Technology Co. Ltd. . All animal experiments were approved by the Drug Safety Evaluation Center of Harbin Medical University, China (1100111084356). The animals were maintained in a specific pathogen–free condition at 26 ± 1°C and 50 ± 5% humidity with a 12-hour light/12-hour dark cycle, and the procedure of animal experiments was performed under the Guidelines for Care and Use of Laboratory Animals of the Drug Safety Evaluation Center of Harbin Medical University (no. SYDW 2020-051). 4T1 tumor models were established by subcutaneously injecting 4T1 cells  with a concentration of 15 mg kg−1. Then, the T1-weighted MR images were obtained before and after 3, 6, 12, and 24 hours of injection.The in vitro and in vivo MR imaging was measured by an animal MR imaging scanner (BioSpec 94/20USR 9.4 T). CVS NPs were dissolved in PBS solution with different Cu concentrations  with or without H−1) through the tail vein. At 0, 3, 6, 12, and 24 hours after injection, the tumor region was imaged using the PA computed tomography system.Female BALB/c mice bearing 4T1 tumors were injected with CVS NPs  as follows: (i) control (PBS), (ii) NIR, (iii) CVS, (iv) CVS + NIR, and (v) CVS + NIR + ice pack (five cycles). Afterward, the body weight and tumor volume were recorded every 2 days, and the tumor volumes (in mm3) were calculated according to V = lw2/2, in which w and l are the width and length of the tumor, respectively. For mice in groups (iii) to (v), CVS NPs  were first injected intravenously, where group (iii) was not further treated and group (iv) was subjected to 808-nm laser (1.0 W cm−2) irradiation at 12, 24, and 48 hours after injection. Group (v) was successively irradiated by an 808-nm laser for 5 min and cooled by an ice pack for five cycles at 12, 24, and 48 hours after injection. Notably, an ice pack was used as the cold source to control the temperature to drop to 15°C.Twenty-five 4T1 tumor–bearing mice were randomly divided into five groups (n = 6) were treated with PBS  or CVS NPs  through intravenous injection. After 1, 7, and 30 days of injection, blood samples were collected. For routine blood analysis and blood biochemistry tests, parameters such as platelets, blood urea nitrogen, mean platelet volume, albumin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, white blood cell, hemoglobin, alanine transaminase, mean corpuscular hemoglobin, alkaline phosphatase, red blood cells, creatinine, and aspartate aminotransferase were measured.Female BALB/c mice without tumors  with Tukey’s multiple comparisons post hoc test was used to compare three or more groups. Quantitative data were indicated as means ± SD. Asterisks were used to represent significant differences . The statistical analysis was performed by using GraphPad Prism 9.0 software.Unpaired two-tailed Student’s


Nannochloropsis gaditana, the bacterium Bacillus safensis, and demonstrated rescue of the microalgae from the pathogen using phage. 16S rRNA amplicon sequencing showed that phage treatment did not alter the composition of the bacteriome. It is widely suspected that the algal bacteriome could play a protective role against bacterial pathogens. To test this, we compared the susceptibility of a bacteriome-attenuated N. gaditana culture challenged with B. safensis to a N. gaditana culture carrying a growth-promoting bacteriome. We showed that the loss of the bacteriome increased the susceptibility of N. gaditana to the pathogen. Transplanting the microalgal bacteriome to the bacteriome-attenuated culture reconstituted the protective effect of the bacteriome. Finally, the success of phage treatment was dependent on the presence of beneficial bacteriome. This study introduces two synergistic countermeasures against bacterial pathogenicity in algal cultures and a tractable model for studying interactions between microalgae, phages, pathogens, and the algae microbiome.The natural assemblage of a symbiotic bacterial microbiome (bacteriome) with microalgae in marine ecosystems is now being investigated as a means to increase algal productivity for industry. When algae are grown in open pond settings, biological contamination causes an estimated 30% loss of the algal crop. Therefore, new crop protection strategies that do not disrupt the native algal bacteriome are needed to produce reliable, high-yield algal biomass. Bacteriophages offer an unexplored solution to treat bacterial pathogenicity in algal cultures because they can eliminate a single species without affecting the bacteriome. To address this, we identified a highly virulent pathogen of the microalga  Microalgal bacterial communities (hereafter referred to as bacteriomes) coexist within the algal phycosphere in most ecosystems  was obtained from Dr. Matthew Posewitz at the Colorado School of Mines in Golden, Colorado. Bacillus safensis was originally identified as Bacillus pumilus by 16S rRNA sequencing  at 25°C, under 215 μmol m−2 s−1 warm white LED light at 4000K color with a 12-h diurnal cycle. Stock cultures were continuously mixed on an orbital shaker at 150 rpm. Initial cultures were seeded into 4-ml F2N•NO3 media from F2N•NO3 plates. The F2N•NO3 media used for subculturing (supplementation of marine broth described below) was F2N•NO3 and consisted of the following components: 34.12 g/L Crystal Sea Oceanic Sea Salt mix, 10 mM Tris HCl, 724 μM (100 mg/L) NaH2PO4•H2O, 25 mM (2.16 g/L) NaNO3, 0.0045 μM (0.76 mg/L) MnSO4•H2O, 0.00015 μM (0.0365 mg/L), Na2MoO4•2H2O, 0.0004 μM (0.115 mg/L) ZnSO2•7 H2O, 0.00025 μM (0.07 mg/L) CoSO4•7H2O, 0.0002 μM (0.034 mg/L) CuCl2 •2H2O, 0.0585 μM (0.023 g/L), Fe(NH4)2(SO4)2•6H2O, 0.0585 μM (0.022 g/L) Na2EDTA•H2O, 1.899 μM (675 μg/L) Cobalamin (B12), 0.51 μM (125 μg/L) Biotin, 8.15 μM (2.75 mg/L) Thiamine HCl. Vitamin stocks were prepared in 50-mM HEPES buffer. F2N•NO3 was filter-sterilized with a 0.2-micron Nalgene Rapid-Flow Sterile Vacuum Filter . Stock cultures were routinely passaged in F2N•NO3 liquid media during mid-log phase growth.N. gaditana was cultivated in 50-ml F2N•NO3 until mid-log phase (~107 cells/mL) in 250-ml baffled flasks before experiments. For the subsequent experiments, 50 ml of subcultures were centrifuged at 7,000 × g for 10 min and resuspended in 20 ml of marine broth-supplemented media to achieve a cell density of ~5 × 106 cells/mL in 125-ml baffled flasks. The marine broth-supplemented media consisted of 1:4 v:v Difco Marine Broth 2216 (BD Diagnostics) and F2N•NO3 (MB/F2N•NO3).Bacillus safensis was inoculated in 4 ml of Luria Broth (LB) and grown overnight at 30°C on an orbital shaker at 200 rpm. The next day, B. safensis was subcultured 1:100 in 50-ml LB media in a baffled flask and incubated at 30°C with aeration to an OD of 1.3 (~8 × 108 CFU/ml). The subculture was transferred to a 50-ml conical tube and centrifuged at 8,000 × g for 7 min at room temperature. The pellet was washed twice in 1:4 v/v MB/F2N•NO3 before diluting to the desired multiplicity of infection (MOI = cell ratio of Bacillus:Nannochloropsis). In total, 100 μl of diluted cells or 100 μl of 1:4 v/v MB/F2N•NO3 media were introduced into 20 ml of cultures seeded with 5 × 106N. gaditana cells.A single colony of 6 plaque-forming units (PFU) with 100 μl of B. safensis culture, inoculated into molten LB agar , and plated onto LB agar plates. After overnight incubation, 5 ml of SM buffer  was added to each plate and agitated by a rotary shaker at room temperature for ~1 h. The SM/top agar mixture was scraped into 50-ml conical tubes, vortexed for 10–15 s, and then centrifuged for 30 min at 5,000 × g, at 4°C. The supernatant was filter sterilized and then titered as described below. To prepare Leo2 for phage treatment experiments, 5–10 ml was dialyzed twice in 2 L of 34 g/L Crystal Sea  using Spectra pre-wetted RC membranes (Repligen part # 132544). After dialysis, the Leo2 suspension was filter sterilized, and 65 μl of phage or sterile crystal sea (control) was added to cultures at a final concentration of ~5 × 107 PFU/ml.Leo2 lysates were produced by mixing ~1 × 10N. gaditana that was passaged once on an antibiotic plate containing 100 μg/ml ampicillin, 30 μg/ml chloramphenicol, 100 μg/ml streptomycin, and 100 μg/ml tetracycline  spontaneously recruited a new microbiome. For each experiment, we confirmed that NgMB− was bacteriome-attenuated by 16S rRNA amplicon sequencing. We also confirmed that NgMB− did not produce bacterial colonies when 10 μl of samples were spread on MB and LB plates, even after >1-week incubation.The bacteriome-intact and bacteriome-attenuated cultures were propagated from a parent culture of N. gaditana cultures grown to ~5 × 107 cells/ml were then sorted into two tubes with a flow rate of ~7,000 cells/min until 5 ml of culture was sorted. Microalgae cells with chlorophyll and aggregate bacterial cells without chlorophyll were collected into two separate tubes. After sorting, separate microalgae and bacterial cells were gently centrifuged  for 5 min, washed, and resuspended in F2N•NO3 twice before final resuspension (below). The transplanted bacteriome cultures were then checked for the correct size and chlorophyll content before mixing with pelleted microbiome-attenuated microalgae from a 5-ml culture to match a similar ratio to the parent culture. The transplanted bacteriome–microalgae mixture was resuspended in 5 ml of F2N•NO3 media and grown at 25°C under 215 μmol m−2 s−1 warm white LED light at 4000 K color with a 12-h diurnal cycle. Subsequently, 10 μl of the cell-sorted bacterial fraction were plated on F2N•NO3 and then grown for 21 days under the conditions described above. No microalgal growth was observed in this medium indicating successful sorting of microalgae from bacteria.The BD FACSAria Fusion cell sorting flow cytometer  was used to sort bacteriome members from the microalgae cells using a 70-μm nozzle at 70 psi. In addition, the 1.0 neutral density filter was inserted into the beam path to allow simultaneous visualization of the whole microalgae and bacteria populations. Subpopulations of the microalgae and bacterial cells were defined by their relative size and microalgal chlorophyll fluorescence in the PE-Cy5  channel. Samples were collected on the days indicated for each experiment. 240 μl aliquots were taken from each flask, with half the volume used to titer bacteria and the remainder used to titer phages (described below). An additional 50 μl of culture was taken from each flask for daily cell counts, and diluted as needed using media. On the indicated days, 667 μl of culture was taken from each flask, and replicates were pooled for DNA extraction and 16S sequencing.i – Wf) – 0.0287, where Wi is the initial weight of 1 ml of water, Wf is the final weight of the water after 5 min of uptake by the cell sorter, and 0.0287 is correcting for the initial uptake of sample inherent to the system. On average, the flow rate was 0.27 μL/s. The microalgal cell density could then be calculated using the following equation: Cell density (cells/ml) = (GE)/(CFR * t), where GE is the number of gated events, CFR is the cytometer flow rate in ml/s, and t is the time in seconds the samples were run.Daily cell counting employed the BD FACSAria Fusion cell sorter using a 70 μm nozzle and a 1.0 neutral density filter. Samples were run for 15 s each, total events were collected, and the microalgae population was gated using the chlorophyll autofluorescence signal detected by the PE-Cy5 channel. To convert between total events and approximate cell density in cells/ml, semi-daily flow rates were measured using water and calculated in ml using the following equation: Cytometer flow rate = . Extracted gDNA was stored at −20°C until processing and outsourcing for 16S sequencing. Samples collected for 16S rRNA gene sequencing were quantitated with either a Qubit 3.0 Fluorometer (Life Sciences) using the High Sensitivity (HS) DNA assay  or a NanoDrop One Fluorospectrometer (ThermoFisher) to quantify the concentration of genomic DNA.Sequencing was conducted by Zymo Research Corporation and their ZymoBIOMIC 16S sequencing service. The ZymoBIOMIC 16S rRNA gene primers  were used to amplify the V3–V4 regions of the 16S rRNA gene. The targeted library preparation, sequencing, and bioinformatics analysis were performed as described previously  according to the manufacturer's instructions, and 500 ng of sample was sequenced by Azenta Life Sciences  using the Illumina MiSeq platform (2 × 250 bp). After demultiplexing, paired-end read files  had 667,491 entries for read 1 and 848,505 entries for read 2. Their assembly was performed using SPAdes v3.9.0 in default mode. Scaffolds below 500 bp were rejected, and a large gap was noted among the remaining scaffolds for coverage values; the 13 scaffolds with coverage of 53 or higher were retained, while the remaining 92 had coverage less than 2, were shorter than 1,300 bp, and were rejected. The final 13-scaffold assembly totaled 3.677 kbp with an N50 of 864564.N. gaditana as a model system based on a recent study showing that B. pumilus inhibits the growth of Nannochloropsis spp.  tested showed reduced algal cell count over time . Bacillus-infected cultures were seeded at the reported multiplicities, the titer of Bacillus in cultures reached ~1 × 108 CFU/ml by day 1. Notably, the titer abruptly declined ~1–3 log on day 2, with reductions proportionate to the starting inoculum, i.e., the lowest MOI showed the greatest decrease  was found for B. safensis, well within the cutoff of 95% ANI that GTDB had set for each of these candidate species. The next highest ANI  was well below this cutoff, and the remaining candidates (including B. pumilis and B. pumilis_N) had ANI values under 92%. These data unambiguously indicated that this isolate was Bacillus safensis. B. safensis has not been reported to be pathogenic to microalgae but is closely related to B. pumilus  were selected to test whether cultures could be rescued by phage Leo2  of the bacteriome were identified; 14 were resolved to the genus level, 3 were identified at the family level , and 1 OTU from Gammaproteobacteria was not identified beyond its class. By OTU counts, the most abundant genera, or “core” members, were Thalassospira, Marinobacter, Oceanicaulis, Robiginitalea, Nitratireductor, Hoeflea, and Sulfitobacter. The less abundant genera were Limnobacter and Methylophaga. B. safensis-infected cultures showed a reduced OTU ratio for Thalassospira and Nitratireductor and an increased ratio for Oceanicaulis relative to the N. gaditana control  treated with phage were similar to the B. safensis OTUs over time relative to the bacteriome members, even in the absence of Leo2  culture. The attenuation was done by passaging N. gaditana once on antibiotics (see Methods section) one year prior to these experiments. Additionally, we used fluorescence-activated cell sorting (FACS) to move the bacteriome from the intact culture and restore it to the attenuated culture. We have denoted the three cultures as NgMB+ (microbiome intact), NgMB− (attenuated), and NgMBTP (transplant)  . The antnsplant) . The thrcteriome . The traNgMB+ and NgMB− cultures with B. safensis at an MOI of 0.001 with and without phage. As expected, NgMB+ and NgMB− crashed after exposure to B. safensis, whereas the uninfected (control) cultures exhibited normal growth  cultures was comparable to that of phage-rescued NgMB+ cultures (B. safensis colonies that were isolated from NgMB− cultures were resistant to Leo2 infection (data not shown).Given the importance of algal bacteriomes shown here and elsewhere  and organic carbon substrates (mixotrophy) , nor phage rescue (treatment of B. safensis-infected cultures) result in extreme shifts in the balance of the bacteriome over time. This indicates that phage Leo2 can rescue pathogen-infected cultures without disrupting the algal bacteriome. Furthermore, the causative agent of B. safensis pathogenicity likely acts on N. gaditana itself rather than inhibiting N. gaditana by altering the bacteriome. The latter point is reinforced by data showing that the causative agent of B. pumilus-mediated inhibition of N. gaditana is the secretion of an active molecule  survived a low-dose B. safensis infection, whereas cultures lacking an intact bacteriome (NgMB−) crashed after the same treatment  culture, which tended to spontaneously recruit bacteria, including algal pathogens. This point emphasizes the need to improve our understanding and use of protective microbiomes as a standard tool for algal cultivation.It would be interesting to test if the bacteriome-imposed growth promotion itself contributes to protection. In principle, strictly protective or strictly growth-promoting members could be distinguished by reintroducing isolates into an axenic algae background and measuring N. gaditana  may provide an opportunity for a deeper study of phage and pathogen interactions within a system in which the health of the host depends on the integrity and syntrophic interaction of a simple and stable microbiome.In addition to a lab-scale proof-of-concept for new crop protection tools, the work presented here introduces a simple model for studying host-microbiome relationships. The complexity, dynamics, and variability of natural microbiomes have led researchers to turn to simplified models to better understand homeostasis and disease-related interactions  can be found in the article/BH: Investigation, Formal analysis, Visualization, Writing—review and editing. MM: Investigation, Formal analysis, Visualization, Writing—original draft. ML: Investigation, Data curation, Formal analysis, Visualization. JS: Investigation, Formal analysis, Writing—review and editing. GL: Investigation, Formal analysis. SK: Investigation, Formal analysis, Writing—review and editing. HB: Investigation, Formal analysis. KW: Investigation, Formal analysis, Writing—review and editing. BR: Resources, Investigation. CS: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing—original draft, Writing—review and editing. JC: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing—original draft, Writing—review and editing.


Saccharomyces cerevisiae, it was previously shown, by a microscopy approach, that Xrn1 is located to different cellular compartments, depending on physiological state. During exponential growth, Xrn1 is distributed in the cytoplasm, while it co-localizes with eisosomes after the post-diauxic shift (PDS). Here, we biochemically characterize the Xrn1-associated complexes in different cellular states. We demonstrate that, after PDS, Xrn1 but not the decapping nor Lsm1-7/Pat1 complexes associates with eisosomal proteins, strengthening the model that sequestration of Xrn1 in eisosomes preserves mRNAs from degradation during PDS.mRNA degradation is one of the main steps of gene expression, and a key player is the 5’-3’ exonuclease Xrn1. In Gene expression is a crucial and well-orchestrated interplay between transcription, translation, and mRNAs and proteins decay. In eukaryotes, cytoplasmic mRNA decay is intimately linked to translation and mediated for a large part by the 5’- 3’ mRNA degradation pathway. After mRNA decapping by the DCP complex, with the assistance of its co-factors, the mRNA is degraded by the exonuclease Xrn1 . This enzyme is highly processive and requires a 5′ monophosphorylated RNA end, produced by decappingS. cerevisiaecells must quickly adapt to survive stress conditions. Consequently, a fast reprogramming of gene expression must be set upGene expression varies, depending on the physiological state of the cells. TheThe eisosome is a structure associated with the plasma membrane near endocytosis sites. The two main components, Pil1 and Lsp1, share 72% sequence identity and are present in around equimolar proportions. However, despite this strong identity, Pil1 appears to be the major actor of eisosome biogenesis . To be efficient, Pil1 and Lsp1 are phosphorylated by two protein kinases, Pkh1 and Pkh2, which are part of the eisosome600 nm0.6 and OD600 nm2), diauxic shift (DS) and Post-diauxic shift (PDS) extracts . LMA5015, Xrn1-TAP:HIS3MX6 and LMA5434, Pil1-TAP:HIS3MX6 are from the TAP collectionpil1∆::KANMX4was constructed by transformation of the LMA5015 strain with a PCR fragment obtained withpil1∆::KANMX4 strain from the Euroscarf collection using oligonucleotide fw: GAATGGACACTAGACTCTGC and oligonucleotide rv: GGGAACAGAAATGATTATCTGTCC. The cell cultures were grown at 30°C in YPGlu medium, up to an OD600nm0.6 or 2.0. To collect the DS and PDS samples, the cultures were diluted to an OD600nm0.005 and grown until they reached the Diauxic State (DS) after 19h of culture or Post Diauxic State (PDS) after 37h of culture. 4000 OD600nmof cell cultures were collected, washed with cold water and stored at -80°C.Affinity purification.The cells were resuspended with lysis buffer  containing a protease-inhibiting reagent (Roche) (1mL per mg of cells). Then, 500 µL of acid-washed glass beads were added per mL of cell suspension and vortexed three-time 40 sec. at 4°C, 6m/sec. . The suspension was centrifuged for 20 min. at 4°C, 4000 rpm. 0.5% Triton was added to the supernatant, followed by 25 µL of covalently coupled IgG-Dynabeads® magnetic beads resuspended in lysis buffer. The lysate was then incubated for 2 hours at 4°C under agitation. The magnetic beads were recovered and washed five times in washing buffer , then once in lysis buffer. They were resuspended in 2% SDS, 1X TE and eluted by incubation at 65°C for 15 min. The SDS was removed with HiPPRTM Detergent Removal Resin kit (Thermo Fisher Scientific). Proteins were precipitated by Methanol/Chloroform method (Wessel and Flügge 1984).Mass spectrometry analysis.The samples were treated as described in . Briefly, endoprotease Lys-C and Trypsin digested peptides were analyzed on an LTQ-Orbitrap Velos mass spectrometer . The raw data were analyzed with MaxQuant (version 2.0.3.0). Only proteins identified with at least 2 peptides were selected for further quantification analysis. The abundance of the identified proteins was achieved in the Perseus environment (version 1.6.14). Protein group LFQ intensities were log2transformed. After comparison with a control group, using the Student’s t test statistic, the results were represented as volcano plots or scatter plots.Data availabilityviathe PRIDE repository. The accession numbers are PXD044514, PXD044631, PXD044634, PXD044653.Raw mass spectrometry data were posted in the ProteomeXchange Consortium10.22002/30gmd-r4w62Description: Data from mass spectrometry analysis. Resource Type: Dataset. DOI:


R in the phosphinate group of ferrocenyl bisphosphinic acids can affect conformational possibilities and coordination packing. It also demonstrates that H-phosphinate plays a key role in variational mobility, while Me- or Ph- substituents of the phosphinate group can only lead to 0D complexes or 1D coordination polymer. Overall, this paper provides valuable insights into the design and construction of coordination polymers based on ferrocene-contained linkers. It sheds light on how different reaction conditions and substituents can affect conformational possibilities and coordination packing, which could have significant implications for developing new polymers with unique properties.This paper illustrates how the size and type of substituent  Recently, metal–organic frameworks (MOFs) have garnered widespread research attention in many fields, including gas separation, energy storage and conversion, wastewater treatment, catalysis, sensors and others ,7,8,9,10While ferrocenes are renowned for their synthetic versatility, the coordination properties of ferrocene-based carboxylic acids have been most extensively studied ,25,26,27This possibility of tuning electronic and steric properties with two aryl/alkyl substituents directly attached to phosphorus atom of phosphinic acids translates into unique properties of materials that cannot be replicated by the utilization of analogous phosphonates or carboxylates ,30,31. TIn acidic environment, polytopic carboxylates MOFs, frequently displaying porosity, often composed of discrete oxometalate secondary building units are bound together by polytopic carboxylate linkers . In the In general, the use of bulky substituents in phosphinate groups can limit the free rotation of the molecule and affect the coordination packing mode in crystals. This is because larger substituents may not allow for complete rotation of the phosphinate group, which can impact on the formation of a three-dimensional supramolecular structure. However, it is important to note that this effect may vary depending on the specific substituent used and other factors such as reaction conditions and solvent. Therefore, careful consideration must be given to these factors when designing and synthesizing coordination polymers and MOFs with specific structural and functional properties .The investigation of the flexibility of bisphosphinic acids was initiated by Midollini et al. This research has contributed to our understanding of the structural properties and behavior of coordination polymers based on alkyl spacers between phosphinate groups. These polymers typically involve divalent metal ions bridged by phosphinate oxygen atoms, and can exhibit a range of structural arrangements, from one-dimensional to three-dimensional architectures. The specific structure formed depends on various factors, such as the nature of the metal used and the reaction conditions employed. By studying these factors, researchers can gain insights into how to design coordination polymers with specific structural and functional properties for various applications ,43.2− anion ligand from chelating a single metal center, due to the increased distance between the two phosphinic groups. This finding has important implications for designing and synthesizing coordination polymers with specific structural and functional properties, particularly in applications where metal chelation is undesirable or needs to be avoided ..1 with t8a contains two different types of ligands in eclipsed conformations characterized by torsion angles of 71° and 75.4°, respectively. One type of ligand binds two samarium ions, forming an eight-membered cycle, while the other participates in stitching such cycles together through a bridging OPO bond, forming a two-dimensional structure  (1) compared to 1,1′-ferrocenediyl-bis(phenylphosphinic acid) (3) leads to the free rotation of the phosphinate groups around the P-C bond, resulting in a bridging type of coordination for 1 and the formation of a coordination polymer 8a. It should be noted that similar coordination polymers based on acid 1 are also formed for Eu(III), Dy(III), and Y(III) derivatives , 184 refined parameters, R(F) = 0.0268, wR(F2) = 0.0718, max (min) residual electron density 0.389 (−0.464) e Å–3.Crystal data for 10: formula C10H16N2NiO4·C12H14FeO4P2·2(H2O), M = 663.01 g/mol, monoclinic, space group P2/c (No. 13), Z = 2, a = 8.8000(18) Å, b = 11.320(2) Å, c = 14.480(3) Å, β = 106.20(3)°, V = 1385.2(5) Å3, ρcalc = 1.590 g·cm–3, μ = 1.374 mm–1, 17,736 reflections collected , θ range = 1.799° to 27.085°, 2418 independent (Rint = 0.0284) and 2393 observed reflections [I ≥ 2σ(I)], 194 refined parameters, R(F) = 0.0328, wR(F2) = 0.0905, max (min) residual electron density 0.531 (−0.300) e Å–3.Crystal data for H-phosphinic) acid 1 (H2fcdHp), 1,1′-ferrocenylenbis(metylphosphinic) acid 2 (H2fcdMep), 1,1′-ferrocenylenbis(phenylphosphinic) acid 3 (H2fcdPhp) were prepared according to procedure mentioned in the literature 2(fcdMep) 2H2OSynthesis of poly[Ni(H (10). Ni(NO3)2·6H2O , 4,4′-bipyridine  in 4 ml DMF/MeOH (1:1) solution was added to H2fcdMep 2  dissolved in DMF/MeOH . The resulting mixture was heated for 12 h at 80 °C, a part of the methanol was evaporated and the several orange crystals of compound was formed, which were suitable for X-ray diffraction analysis.1–3 with M(II) and M(III) ions. The results demonstrate that the type of acid and metal ion used significantly affect the resulting polymer’s structure and properties. Our study emphasizes the crucial role of ligand design in regulating the structure and properties of coordination polymers.In this paper we summarized the synthesis of coordination compounds and MOFs based on ferrocenylbisphosphinic acids H-phosphinate plays a key role in variational mobility, while Me- or Ph-substituents of the phosphinate group can only lead to 0D complexes or 1D coordination polymers. With the use of additional ligands, such as 4,4′-bipyridine, the formation of a 3D structure is possible, but in this case, the use of H-phosphinate is necessary since the size of the substituent at the phosphorus atom has a significant effect on the formation of structures with different architectures. For acid 1, a bridging type of coordination is observed due to the free rotation of the phosphinate groups around the P–C bond. Thus, this study highlights the importance of careful ligand design to control the structure and properties of coordination polymers.It was demonstrated that However, further research is required to fully explore their potential in this area. The conclusions drawn in this paper will assist researchers in the design and synthesis of coordination polymers and MOFs in the future.


Emulsions have been widely used as immunological adjuvants. But the use of materials derived from plants such as cottonseed oil, alpha-tocopherol, or minerals such as zinc, as well as their use at the nanometric scale has been little explored. In this study, we develop a new miniemulsion and evaluated its antioxidant and phagocytic capacity, as well as parameters related to immune response stimulation by cytokine expression and antibodies production in a mice model.Formulated CN (cottonseed oil miniemulsion) and CNZ (cottonseed oil miniemulsion whit zinc oxide nanoparticles) miniemulsions were characterized by scanning electronic microscopy SEM, DLS and FT-IR. In murine macrophages, splenocytes and thymocytes primary cultures safety and cytotoxicity were determined by MTT. In macrophages the antioxidant and phagocytic capacity was evaluated. In BALB/c mice, the stimulation of the immune system was determined by the expression of cytokines and the production of antibodies.p = 0.108), splenocytes (p = 0.413), and thymocytes (p = 0.923). All CN and CNZ doses tested induced nitric oxide and antioxidants production in dose dependent manner when compared with control. CN-ovalbumin and CNZ-ovalbumin treatments in femoral subcutaneous tissue area showed inflammation with higher leukocyte infiltration compared with FCA. The intraperitoneal administration with CN, CNZ, and FCA showed a higher total intraperitoneal cells recruitment (CD14+) after 24 h of inoculation than control (p = 0.0001). CN and CNZ increased the phagocyte capacity with respect to untreated macrophages in the Candida albicans-phagocytosis assay. The evaluation of residual CFU indicated that only CN significantly decreased (p = 0.004) this value at 3 h. By other side, only CN increased (p = 0.002) the nitric oxide production. CNZ stimulated a major INFγ secretion compared with FCA at day 7. A major IL-2 secretion was observed at days 7 and 14, stimulated with CN and CNZ. Both miniemulsions did not affect the antibody isotypes production  at days 7, 14, 28, and 42. CN induced a significant IgG production against OVA, but lesser than FCA.The CN and CNZ presented stability for 90 days. Immediately after preparation, the CN presented a higher particle size (543.1 nm) than CNZ (320 nm). FT-IR demonstrated the correct nanoparticle synthesis by the absence of sulfate groups. CN and CNZ (1.25 to 10 µL/mL) had no toxic effect on macrophages (The two new miniemulsions with adjuvant and antioxidant capacity, were capable of generating leukocyte infiltration and increased cytokines and antibodies production. Adjuvants have been used to increase the antigens immunogenicity in vaccines or to modulate the immune system in diverse conditions of disease. Adjuvants can be obtained from plants, bacterial products, complex carbohydrates, animal extracts, and synthetic cytokines . EmulsioSqualene is a type of unsaponifiable lipid that acts as a biosynthetic precursor to all steroids in plants and animals . Some em4 • H2O), sodium hydroxide (NaOH), Tween 20, and DMSO (dimethyl sulfoxide) were purchased from Sigma-Aldrich Inc . Fetal bovine serum (FBS) , DMEM culture medium , 3- bromide (MTT) , the Antioxidant assay kit , Griess reaction , Lipopolysaccharide , Neutral Red Assay Kit-Cell Viability/Cytotoxicity , Phagocytosis assay kit (IgG FITC) , C. albicans (ATCC 10261). Ovalbumin (OVA), , Freund’s Adjuvant Complete (FCA), . Ig Isotyping Mouse Uncoated ELISA kit . Mouse Th1/Th2/Th17 Cytokine kit . Formaldehyde . Hematoxylin and eosin (H & E) , CD14 Monoclonal Antibody (Sa2-8), APC, eBioscience .All reactive were used without additional purification, cottonseed oil pharmaceutical grade, α-tocopherol (96% purity), zinc sulfate monohydrate (ZnSO4 • H2O) was dissolved in deionized water at 0.5 M that remained under stirring at 300 rpm while sodium hydroxide solution (2 M NaOH) was added in drops. After, the supernatant was discarded, and the precipitate formed was washed with deionized water and centrifuged at 8,000 rpm for 10 min. Precipitate was dried in a hot air oven at 60 °C for 24 h. Then, the obtained powder was calcinated at 300 °C for 2 h, in a muffle.The zinc oxide (ZnO) nanoparticles were obtained by the coprecipitation method. Zinc sulfate monohydrate .Two miniemulsions were produced as follow: (1) cottonseed oil miniemulsion (CN) (to form the oil phase of 275 mg), in which α-tocopherol (25 mg), Tween 20 (35 mg) and DMSO (50 ml) were dissolved, and (2) cottonseed oil miniemulsion + ZnO nanoparticles (CNZ) was formulated in the same way as described above plus the addition of ZnO nanoparticles (2.5 µg) in the oil phase. The oil phase was calibrated with saline solution (1 mL) and mixed at 12,000 rpm in homogenizer for 10 min  were determined by dynamic light scattering (DLS) and zeta potential (PZ) was determined by electrophoretic mobility measurements in a ZS90-Nano Zetasizer . All results were measured in triplicate at room temperature (25 °C). The ZnO nanoparticles were characterized by X-ray diffraction (XRD) and the Fourier Transform Infrared Spectroscopy (FT-IR), ZnO nanoparticles morphology and size were confirmed by scanning electron microscopy (SEM), and the droplet size was confirmed by atomic force microscopy (AFM), images were analyzed with Gwyddion software. The characterization of miniemulsions functional groups and their components  were determinate by attenuated total reflection (ATR) in conjunction with FT-IR technique. The samples were placed onto the diamond ATR crystal, and spectra was recorded by a FT-IR , with an accumulation of 16 scans at a resolution of 4 cmin vivo and ex vivo experiments. The animals were obtained from the Bioterium of Facultad de Ciencias Biológicas of the Universidad Autónoma de Nuevo León,  under the approval of Bioethics committee protocol No. CEIBA-2019-015. Animals were housed eight per cage (polypropylene) under constant specific conditions with temperature at 24 °C, 50% relative humidity and a controlled light and dark cycle (12 h:12 h) with ventilated caging systems, wood sawdust bedding and standardized environmental enrichment. Feed  and clean water were supplied ad libitum. For sample  collection, animals were euthanized by overdoses of anesthesia (ketamine-xylazine protocol).Male BALB/c mice  were used for all 2 and 95% air at a constant temperature of 37 °C.Thymus, spleen, and peritoneal macrophages primary cultures were obtained from healthy mice. Cell cultures were maintained in a medium containing 5% fetal bovine serum (FBS), antibiotic and antimycotic 1%, under an atmosphere of 5% COAll the experiments pertaining to animal use and their care strictly followed the guidelines of the Official Mexican Standard (NOM-062-ZOO-1999) on technical specifications for production, care and use of laboratory animals. All the protocols were approved by UANL FCB Animals Ethics Committee (protocol CEIBA-2019-015).in vitro on thymus, spleen, and peritoneal macrophages primary cultures. Cells were seeded in 96-well plates at a density of 2 × 105 cells/well with 100 µL of DMEM culture medium. After 24 h of incubation culture medium was replaced by new one containing CN or CNZ . Cell viability was evaluated 24 h after treatment using the technique of MTT.The miniemulsions toxicity was evaluated The spectrophotometry measurement was performed in triplicate at 570 nm in a plate reader , the percentage of viability was determined by the following equation:For the miniemulsions effect over nitric oxide production (as nitrite) and total antioxidant capacity (TAC), murine peritoneal macrophages (MPM) were used. As positive control lipopolysaccharide (LPS) was used in a concertation of 5 µL/mL .Nitrite concentration in treated cells supernatant was determined by Griess reaction . MPM were cultured in the same way as mentioned in the cell toxicity assays section described above. Supernatants (50 μL) were collected, centrifuged at 600× g for 5 min, mixed with 100 μL of Griess reagent (0.1% N-(1-naphthyl)-ethylene diamine, 1% sulfanilamide in 5% phosphoric acid) and incubated at room temperature for 10 min. Absorbance was measured at 540 nm .+2 to Cu+ observed by a colored complex formation. Trolox  standard curve was measured at 540 nm.TAC was determinate in MPM culture supernatants. MPM were cultured and supernatants were collected under conditions described above. The Antioxidant assay kit  was used according to manufacturer’s instructions. Assay measures TAC by the reduction of Cu5 murine peritoneal macrophages were seeded on 96-well plates, washed three times with PBS, and then incubated with 10% neutral red for 2 h. After three washes, neutral red incorporated into lysosomes was dissolved by the lysis solution provided by the kit, and the absorbance was read at 540 nm. Qualitative phagocytic activity was determined using Phagocytosis assay kit (IgG FITC) . MPM were seeded on coverslips in six-well plates and incubated for 24 h in DMEM medium. After 1 h of treatments cells were incubated with latex beads-rabbit IgG-FITC complex for 2 h. Photographies were observed in fluorescence microscope .Two assays were designed to determine the miniemulsions uptake and phagocytic activity effect in MPM. CN and CNZ (10 µL/mL) were used as cell stimulation treatment, for phagocytosis inhibition control disodium clodronate (0.6 mg/ mL) was used and DMEM medium as negative control. MPM quantitative uptake capacity was determinate by neutral red assay  using thC. albicans (ATCC 10261) was used. MPM, obtained from three healthy mice were seeded under the previously described conditions in the cell toxicity assay. Phagocytosis assay was performed based in the method proposed by 6 cells)/C. albicans (1 × 106–1 × 108 cells) proportion) was collected to assess the increase or decrease of colony forming units (CFU) by the Mc-Farland method  production was also determined as mentioned previously.Death of Three independent experiments were designed. Ovalbumin (OVA),  was used as antigen . All immunizations were subcutaneously applied (s.c.) in a total volume of 100 µL per animal (50 µL of miniemulsion and 50 µL of antigen). Freund’s Adjuvant Complete  was used as positive control; a group of only antigens was used as control .+ expression was determined by flow cytometry. These assays were carried out by flow cytometry .Experiment A. Mice inoculated with CN, CNZ, FCA and control were sacrificed by cervical dislocation at 24 h post-treatment. The administration site  was fixed in 10% formaldehyde solution and after that, covered with hematoxylin and eosin (H & E) to determine leukocyte infiltration. For the quantification and cell typing, mice were treated intraperitoneally with miniemulsions without antigen. The cells (cells/µL) were located by size and granularity to stablish the gate, and the CD14Experiment B. Mice were immunized with OVA, and its combination with CN, CNZ, and FCA or control  on days 0 and 14. Blood samples were taken on days 7, 14, 28, and 42. The blood samples were obtained from cardiac punction in anesthetized mice. Blood serum was separated and frozen at −80 °C until the time of isotype antibody determination  and determination of cytokine profile TH1 /TH2 /TH17 (IL 71a). Cytokines were measured by flow cytometry  using a commercial Cytometric Bead Array (CBA) Mouse Th1/Th2/Th17 Cytokine kit .Experiment C. Mice were immunized at days 0 and 14 with the different miniemulsions (CN or CNZ) previously mentioned and challenged with OVA at day 42 and, blood samples were taken at 72 h after. The blood serum was separated to evaluate specific antibodies and frozen at −80 °C until analysis.2SO4. Plate was read at 450 nm in a plate reader , values indicating optical density (DO). The antibodies isotypes  were determinate by commercial kit Ig Isotyping Mouse Uncoated ELISA .Anti-ovalbumin antibodies were evaluated by ELISA, as previously described . Briefly® (2014) software. Data was expressed as the mean ± standard error (SE). For the variables of nitric oxide production and antioxidant capacity, a Pearson correlation test was performed. Statistical significance (α = 0.05) was determined by ANOVA. The comparison of means was performed by Tukey test.All data analysis was performed with MinitabThe miniemulsion CN and CNZ presented stability for 90 days, and this was confirmed by DLS analysis  and 1B. 4 • H2O and ZnO nanoparticles is shown in 4 • H2O, a hydroxyl group (OH) at 3,135 cm−1, a carbonyl group (C=O) at 1,506 cm−1, and sulfate groups (SO−24) at 1,090 y 855 cm−1. The spectra of ZnO nanoparticles  (The FT-IR spectra of ZnSOarticles  showed apy (SEM)  showed aThere is a qualitative difference between two samples in the size of the emulsions, considering that in −1; stretching vibrations of carboxyl group (COOH) at 2,856 cm−1, ketone group (C=O) at 1,744 cm−1, and C-O group at 1,156 cm−1. The spectra of α-tocopherol , at 1,461 cm−1 for an asymmetric bending of a phenyl group (C6H5), and methyl and methylene symmetric bending peaks at 1,365 and 1,203 cm−1, respectively.The copherol  showed p6H5 functional groups of α-tocopherol , splenocytes (p = 0.413), and thymocytes (p = 0.923) in vitro (p = 0.998) and 2.5 (p = 0.112) µL/mL and highest at doses of 5 and 10 µL/mL (p = 0.0001) when compared with the positive control (LPS). No difference (p = 0.935) in nitric oxide production was found at all doses tested of CNZ when compared with LPS (CN and CNZ miniemulsions at concentrations of 1.25 to 10 µL/mL had no effect on the viability of macrophages (in vitro . All doswith LPS .p = 0.0001). The CN 5–10 µL/mL doses induced a major antioxidant production (p = 0.0001) than LPS and 2.5 µL/mL (p = 0.979)/1.25 µL/mL (p = 1.0) doses induced similar productions levels. The CNZ treatment at 10 µL/mL (p = 0.002) dose induced a higher antioxidant total production than LPS, but at minor doses 5 µL/mL (p = 0.115), 2.5 µL/mL (p = 0.925) and 1.25 µL/mL (p = 1.0) no difference was found (p = 0.0001) as in those treated with CNZ .All CN and CNZ doses tested had the capacity to induce the production of antioxidants by dose dependent manner when compared with the control , however no difference was found between CN, CNZ and FCA  with CN or CNZ with ovalbumin showed inflammation with higher leukocyte infiltration compared with CFA . The int and FCA .p = 0.158) and CNZ (p = 0.495) miniemulsions evaluated by neutral red assay did not affect the macrophages uptake activity when compared with untreated macrophages  this value respect CNZ, but at 4 h no difference was found with respect to the untreated macrophages (p = 0.002) the nitric oxide production in all times evaluated , this difference was not observed at day 14 (p = 1.0) (p = 0.027) and 14 (p = 0.007), stimulated with the CN and CNZ miniemulsions (The CNZ miniemulsion stimulated a major IFNγ secretion compared with FCA at day 7 (p = 1.0) . A majormulsions . Howevermulsions .p = 0.044) (p = 0.044) was found with FCA at day 28  . The CN t day 28 . At the p = 0.0001)  .The droplet size obtained from CN (543.1 nm) and CNZ (320 nm) formulations, the zeta potential (−26.6 mV for CN and −28.1 mV for CNZ) and PDI values (0.074 for CN and 0.449 for CNZ) indicate a monodisperse emulsion with a greater molecule repulsive strength which increases its stability .−1, characteristic of the ZnO stretching mode    , and α-t groups) .The charges detected by Z potential measurements can be attributed to the unsaturated fatty acids group of cottonseed oil, these molecules have a negative charge . In factThe miniemulsion with ZnO nanoparticles showed more stability  due to the interaction of solid particles with the oil/water interface, this effect increases the stability by steric barrier, this is known as a Pickering stabilizer .In our study the stimulation with CN and CNZ miniemulsions could activate the nitric oxide production in macrophages, indicating their activation. This activation is similar when macrophages are challenge with antigens, adjuvants, or vaccines, inducing nitric oxide production and the capacity to increase the immune response . The proThe antioxidant total production capacity was studied, determining that both miniemulsions have the capacity to induce higher levels of antioxidants, that can be correlated with the promotion of mitochondrial biogenesis increasing antioxidant enzymes production, decreasing the exacerbation of proinflammatory cytokine response, and blocking or scavenging the excessive response of ROS and RSN, that in higher amounts can affect a healthy immune response . Is impo+ after intraperitoneal miniemulsions injection, similar to the MF59 adjuvant, that is an oil-in-water emulsion containing squalene (4.3%) in citric acid buffer with stabilizing non-ionic surfactants Tween 80 (0.5%) and Span 85 (0.5%) used in vaccines  and the purified saponin QS-21) in a liposome-based formulation) or AS03  emulsion). In addition, the production of IFN-γ could be mediated by IL-2 produced by vaccine-induced effector memory T cells (Several studies report that IFN-γ derived from innate immune cells enhances protective antiparasitic Th1 responses and malaria protection after use of adjuvant AS01 (containing two immunostimulants (TLR4 ligand 3- T cells . Increas+ T cell dependency (+ deficient mice, and to stablish the dependence of Th1 or Th2.Only CN miniemulsion induced a significant OVA specific IgG production, however no IgG isotype antibodies switch was observed. This can suggest the lack of CD4pendency . HoweverTM after a single immunization (The detection of IgG at day 45 can suggest a sustained immunity induced by lymphocyte B and T using CN miniemulsion. The fact of finding a higher IgG concentration induced by FCA than CN miniemulsion can be attributed to the formation of FCA depots at the injection site, inducing delayed germinal center formation promoting higher antibody responses than a non-depot- inducing adjuvant. Such as MF59-like squalene emulsion AddaVaxnization . These rTM. Oils, such as olive, rice bran, corn, peanut, rapeseed, sunflower, and cottonseed contain 0.1 to 4 g/Kg of squalene (According to our results, we suggest that our miniemulsions have mechanisms of action similar to MF59-like squalene emulsion AddaVaxsqualene . Commercsqualene . If compsqualene .In this study, we synthesized two new miniemulsions cottonseed oil-based, containing or not ZnO nanoparticles with adjuvant and antioxidant capacity, capable of generating leukocyte infiltration and increase cytokines and antibodies production. However, it is necessary to carry out evaluations to determine its effect on the immune system with various antigens and species.10.7717/peerj.14981/supp-1Supplemental Information 1ELISAs and flow cytometry.Click here for additional data file.


In myocardial infarction, a blockage in one of the coronary arteries leads to ischemic conditions in the left ventricle of the myocardium and, therefore, to significant death of contractile cardiac cells. This process leads to the formation of scar tissue, which reduces heart functionality. Cardiac tissue engineering is an interdisciplinary technology that treats the injured myocardium and improves its functionality. However, in many cases, mainly when employing injectable hydrogels, the treatment may be partial because it does not fully cover the diseased area and, therefore, may not be effective and even cause conduction disorders. Here, we report a hybrid nanocomposite material composed of gold nanoparticles and an extracellular matrix-based hydrogel. Such a hybrid hydrogel could support cardiac cell growth and promote cardiac tissue assembly. After injection of the hybrid material into the diseased area of the heart, it could be efficiently imaged by magnetic resonance imaging (MRI). Furthermore, as the scar tissue could also be detected by MRI, a distinction between the diseased area and the treatment could be made, providing information about the ability of the hydrogel to cover the scar. We envision that such a nanocomposite hydrogel may improve the accuracy of tissue engineering treatment. Cardiovascular diseases are a major cause of morbidity and mortality in developing countries ,2, and mIn cardiac tissue engineering, functional cardiac patches are composed of the contracting cells of the heart and extracellular matrix (ECM)-mimicking materials. The ECM provides structural and functional support to the injected cardiac cells, allowing cell reorganization into a functional heart patch and maintaining their viability after transplantation. Such cardiac cell-containing hydrogels were injected directly into the diseased area of the heart to repopulate the injured myocardium and restore its function ,12,13,14Over the past years, several studies have demonstrated the ability of conductive nanostructures, particularly gold nanoparticles (AuNPs), to improve the properties of the engineered tissue ,16. We pHere, we demonstrate, for the first time, the ability of the injectable nanocomposite hydrogels to accumulate within the diseased area of the heart after myocardial infarction, allowing to accurately image the treatment in relation to the actual scar tissue. Several studies have shown the use of functionalized AuNPs with gadolinium chelates to serve as a promising theranostic agent for magnetic resonance imaging (MRI) ,18,19. Hw/v) HAuCl4  was mixed with 20 mL of purified water and heated to boiling. Next, 404 µL of trisodium citrate  was added under stirring. A few minutes later, the color of the solution changed from clear yellow to deep red. After the solution was cooled to room temperature, it was centrifuged at 12,000 RPM for 20 min to precipitate the nanoparticles.Spherical AuNPs were synthesized using sodium citrate according to the methodology described by Enüstun and Turkevich . Briefly2O3 in water for 6 h under reflux led to the formation of GdL as a white solid. Coating of AuNPs with GdL was accomplished through the direct addition of an aqueous solution of GdL to a solution of AuNPs [Au-GdL NPs were prepared according to the method described by Ji-Ae Park et al.  with sliof AuNPs . StirrinThe size, shape, and uniformity of AuNPs and Gd-AuNPs were measured using transmission electron microscopy (TEM) . Samples were prepared by dropping 5 μL of NP solution on a copper grid and then left to dry overnight before imaging. Contrast images were acquired in TEM and captured using SIS Megaview III and iTEM . For Gd-AuNPs, negative staining using uranyl acetate was used for AuNP and Gd-AuNP samples. The absorption spectrum of AuNPs and Gd-AuNPs was evaluated using an ultraviolet–visible (UV/Vis) spectrophotometer . The size and charge of AuNPs and Gd-AuNPs were tested using dynamic light scattering (DLS), and their zeta potential was determined . Furthermore, Fourier-transform infrared spectroscopy (FTIR)  analysis was performed for all synthesis steps: AuNPs, DTPA complex, GdL complex, and Gd-AuNPs.−3 M Tris, 5 × 10−3 M ethylenediaminetetraacetic acid (EDTA) and 1 × 10−6 M phenylmethanesulfonyl-fluoride, pH 8.0) for 1 h. Next, tissues were frozen and thawed three times in the hypotonic buffer. After that, tissues were washed gradually with 70% (v/v) ethanol and 100% ethanol for 30 min each. Next, lipids were extracted using three washes of 100% acetone, 30 min each, followed by a 24 h incubation in a 60/40 (v/v) hexane/acetone solution (solution was changed three times). Then, the tissue was washed in 100% ethanol for 30 min and incubated overnight at 4 °C in 70% ethanol. Next, the tissue was washed with PBS (pH 7.4) four times and incubated in 0.25% Trypsin-EDTA solution  overnight. Then, the tissue was washed with PBS and incubated in 1.5 M NaCl for 24 h (solution was changed three times), followed by washing in 50 × 10−3 M Tris (pH 8.0) and 1% Triton X-100 (Sigma-Aldrich) solution for 1 h. The tissue was washed in PBS followed by double-distilled water, and then frozen (−20 °C) and lyophilized. The dry, decellularized omentum was ground into powder . The omentum was then enzymatically digested under stirring for 96 h at room temperature, in a 1 mg·mL−1 solution of pepsin  in 0.1 M HCl. Next, the pH of the decellularized omentum was adjusted to 7.4 using DMEM/F12 × 10  and 5 M NaOH, to reach final concentration of 1% (w/v).Omenta were decellularized as previously described . BrieflySamples were mounted onto aluminum stubs with conductive paint. High-resolution images were obtained using an HRSEM ZEISS Gemini 300.Gd-AuNP implants were imaged after mounting onto aluminum stubs with conductive paint and without additional coating using a X-Flash 6/60 Bruker HRSEM with a field-emission gun (FEG) electron source. Imaging was carried out under low vacuum with a high voltage of 20 kV and a working distance of 7.3 mm.−1 and a strain of 1%.Rheological measurements (n = 6) were performed using a Discovery HR-3 hybrid Rheometer  with 8 mm diameter parallel plate geometry and a Peltier plate to maintain the sample temperature. Samples were prepared by encapsulation of AuNPs or Gd-AuNPs, to reach a final hydrogel percentage of 0.2%. Samples were loaded at a temperature of 4 °C, which was then raised to 37 °C, to examine the solidification process. The oscillatory moduli of samples were tested at a fixed frequency of 0.8 rad·s−1 in DMEM/F12 . Cells were maintained in NutriStem™  medium containing 0.1% penicillin/streptomycin  and cultured under a humidified atmosphere at 37 °C with 5% CO2. The medium was refreshed daily, and cells were passaged at 70% confluence by treatment with 1 mL of ReLeSR™ .iPSCs were generated from omental stromal cells and were a kind gift from Dr. Rivka Ofir from Ben Gurion University. The undifferentiated cells were cultivated on 10 cm culture plates precoated with Matrigel™  diluted to 250 µg·mL4·5 H2O, 0.5 mM ZnSO4·7 H2O, and 1.5 mM Vitamin B12 . This medium was refreshed every other day.Prior to differentiation, cells were dissociated with Accutase™  and passaged to six-well plates. Until 100% confluence of iPSCs, NutriStem™  was refreshed. On day 0, the medium was changed to 3 mL of RPMI , supplemented with 0.5% L-glutamine , B27-Insulin  and 4.5 µM CHIR-99021 . On day 2, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine, B27-insulin, and 5 µM IWP-2 (Tocris). On day 4, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27-insulin. This medium was refreshed on day 6. On day 8, the medium was changed to 3 mL of RPMI supplemented with 0.5% L-glutamine and B27, and this medium was refreshed on day 10. From day 12, the medium was changed to M-199 , supplemented with 0.1% penicillin/streptomycin, 5% fetal bovine serum , 0.6 mM CuSOw/v pristine hydrogel to achieve a final hydrogel concentration of 0.6%. For control implants, the hydrogel was diluted with 5% FBS–M199 medium at the same ratio.Gd-AuNPs at a concentration of 0.23 M were synthesized as previously described. Then, Gd-AuNPs were encapsulated within 1% −1, Sigma-Aldrich) and propidium iodide  for 20 min at 37 °C. Live and dead cells within the different implants were visualized by an inverted fluorescence microscope , 1 week after the encapsulation.Implant viability was determined using a live/dead fluorescent staining assay with fluorescein diacetate (7 µg·mLv/v) Triton X-100 for 10 min and blocked with 5% bovine serum albumin in PBS for 1 h. Then, implants were stained with primary antibody in 0.5% blocking solution for 2 h, with α-sarcomeric actinin . After three washes with 0.5% blocking solution, implants were incubated with Alexa Fluor 647-conjugated goat anti-mouse antibody  in 0.5% blocking solution for 2 h. For nucleus detection, implants were incubated for 5 min with Hoechst 33258  in PBS and washed three times. Samples were imaged using a confocal microscope (Nikon Eclipse NI-E). Images were processed and analyzed using NIS elements software BR 3.2 (Nikon Instruments).Implants were fixed in 4% formaldehyde for 20 min, permeabilized with 0.1% .iPSCs-derived cardiomyocyte implants were exposed to 1 µM norepinephrine  solution, and then diluted in HBSS medium for 30 min, in 37 °C. The implants contractions were acquired before and after the exposure to noradrenaline at the same position and magnification, using an inverted fluorescence microscope and ORCA-Flash 4.0 digital complementary metal–oxide semiconductor camera  at 100 frames·s−1. The following sequence parameters were used: repetition time (TR) = 4.5 ms; echo time (TE) = 2.3 ms; flip angle (FA) = 10°; field of view (FOV) = 25 × 25 mm2; matrix size = 192 × 192; slice thickness = 1 mm.AuNP and Gd-AuNP solutions, the composite hydrogel, pristine hydrogel, and water for control were imaged using a 7T/30 MRI Biospec  equipped with a gradient unit of 660 mT·mPermission was granted by the Institutional Animal Care and Use Committee (IACUC) of Tel Aviv University, protocol number 04-20-012—“Treatment of biological hydrogel and cardiac cells on cardiac regeneration after ischemia–reperfusion injury in mice model”.IRI was induced in C57BL/6 12 week old male mice. Mice were anesthetized with 3% isoflurene, intubated, and ventilated. After the opening of the chest by a left thoracotomy, the major coronary artery was occluded with 0–8 Prolene sutures  for 45 min. Then, 25 µL of the treatments were injected directly onto the scar tissue . Treatment groups were saline, pristine hydrogel, and composite hydrogels including AuNPs or a 1:1 ratio of AuNPs and Gd-AuNPs. Hydrogels were injected in their solution form at 4 °C, with a final concentration of 0.2%. Then, coronary artery ligation was removed. MRI imaging was performed before the surgery, as well as 1 and 6 weeks post surgery. Mice were sacrificed at the end of the experiment, and their hearts were removed for further analysis.−1. MRI scans were acquired using a cross coil setup including an 86 mm resonator and a 20 mm array of surface coil. The body temperature of the animals was maintained by circulating warm water, and all MRI scans were performed with simultaneous ECG and respiration gating. Mice were placed in the magnet with the ears positioned at the isocenter. The MRI protocol included delayed contrast enhancement achieved by intravenous injection of 0.75 mmol·kg−1 Gd-DTPA  following the initial baseline scanning. In order to identify the morphology of the heart tissue, Cine-FLASH (fast low angle shot) MRI was used to image the heart in a short-axis and long-axis view. Six short-axis slices covering the heart from the base to the apex and one long-axis slice were obtained to evaluate myocardial function and infarct size. The following sequence parameters were used: repetition time (TR) = 4.5 ms; echo time (TE) = 2.3 ms; flip angle (FA) = 10°; field of view (FOV) = 25 × 25 mm2; matrix size = 192 × 192; slice thickness = 1 mm. Cine movies with 10 frames per slice were reconstructed. Each Cine scan lasted 2 min. In order to identify the scar tissue and the location of Gd-AuNPs within the scar tissue, Cine-FLASH  = 8 ms; echo time (TE) = 3 ms; flip angle (FA) = 25°; field of view (FOV) = 25 × 25 mm2; matrix size = 192 × 192; slice thickness = 1 mm. Cine movies with 10 frames per slice were reconstructed. Each CINE scan lasted 2 min.C57BL/6 male mice, weighing from 24 to 32 g, were examined before, as well as 1 and 6 weeks after, the IRI. All experimental procedures were performed under general anesthesia induced by 1–1.5% isoflurane in pure oxygen. MRI imaging was performed using a 7T/30 MRI Biospec  equipped with a gradient unit of 660 mT·mImage processing analysis was performed on the MRI images. First, the location of Gd-AuNPs was assessed by a segmentation process using the K-means algorithm. In this process, the MRI image was divided into four regions with similar gray values. The AuNP area was labeled in yellow. Next, in order to determine the scar area, image enhancement was improved by image subtraction, where a non-contrast image was subtracted from each post-contrast image. A Despeckle filter was then used to eliminate small areas of noise. The scar area was labeled in red. All measurements were performed in ImageJ, on an end-diastolic volume frame within the same MRI slice.AuNPs were synthesized as previously described . The parTo form the composite hydrogel, Gd-AuNPs were encapsulated within the ECM-based hydrogel to interact with the polymeric backbone. The composite hydrogel was thermoresponsive and formed a gel after heating to 37 °C a. High-rThe mechanical properties of the nanocomposite hydrogel are extremely important for cell culture and tissue formation ,26,27,28Next, we tested the biological effect of the Gd-AuNPs on iPSCs-derived cardiomyocytes. The viability of cardiac cells encapsulated within the nanocomposite hydrogel was assessed by a live/dead assay, revealing high viability as within the pristine hydrogel a,b, indiWe next sought to examine the suitability of the nanocomposite hydrogel for MRI imaging. Commonly, high signal intensity is observed after a Gd injection into scar tissue. However, it was previously found that, at high concentrations (above 0.03 M), Gd results in a hypointense signal ,30,31. ANext, the Gd-conjugated AuNP nanocomposite hydrogel, nanocomposite hydrogel without Gd coating, pristine hydrogel (without NPS), or saline was intracardially injected into the LV following an IRI. The mice were imaged 1, 4, and 6 weeks post injection by a 7 T animal MRI scan. To analyze the location of the composite hydrogel in relation to the scar tissue within the LV wall, image processing was used. For the detection of the composite hydrogel, we used the segmentation process . In this process, the MRI image was divided into regions with similar gray values. The area of the nanocomposite hydrogel was easily detected and labeled in yellow a–c. The