Thymus vulgaris L. Biomass yields and amounts of terpenoids were examined, and the lights’ productivity and electrical efficiency were determined. All results were compared to two conventionally used light fixture types (high-pressure sodium lamps (HPS) and fluorescent lights (FL)) under naturally low irradiation conditions during fall and winter in Berlin, Germany. Under LED, development of Thymus vulgaris L. was highly accelerated resulting in distinct fresh yield increases per square meter by 43% and 82.4% compared to HPS and FL, respectively. Dry yields per square meter also increased by 43.1% and 88.6% under LED compared to the HPS and FL lighting systems. While composition of terpenoids remained unaffected, their quantity per gram of leaf dry matter significantly increased under LED and HPS as compared to FL. Further, the power consumption calculations revealed energy savings of 31.3% and 20.1% for LED and FL, respectively, compared to HPS. In conclusion, the implementation of a broad-spectrum LED system has tremendous potential for increasing quantity and quality of Thymus vulgaris L. during naturally insufficient light conditions while significantly reducing energy consumption.A light-emitting diode (LED) system covering plant-receptive wavebands from ultraviolet to far-red radiation  was investigated for greenhouse productions of  Although most common horticultural crops depend on daily light integrals (DLIs) of 6 to 50 mol mr months ,4,5. Ther months . Hence, r months .Today, light-emitting diodes (LEDs) have the potential to replace traditional light sources such as high-pressure sodium lamps (HPS) ,10 and fHowever, the majority of LED radiation studies on plant development have only included narrow wavebands of red (R) and blue (B) light, as these wavelengths are maximally absorbed by the plant’s light-capturing chlorophylls . InitialRecently, studies have suggested further photosynthetic improvements by adding far-red (FR) wavelengths to R spectra, for example, increasing FR radiations promoted growth of seedlings by increasing leaf expansion and whole-plant net assimilation, decreased anthocyanins and carotenoids, and reduced antioxidant potentials ,24,25.As recent studies confirm, green (G) light can also contribute to plant development and growth ,27,28. EDue to the multitude of photobiological studies conducted, it is now well established that wavelengths between ~ 360 and 760 nm influence plants’ photosynthesis, physiology, morphogenesis, and phytochemical contents [Nevertheless, negative side effects resulting from narrow waveband LED applications, such as unwanted photomorphogenic and physiological disorders, pest and disease pressures, as well as difficult visual assessment of plant-status absent under  broad light spectra, have to be further minimized ,35.In consequence, LED fixtures with broader spectral quality covering the range of the photosynthetically active radiation (PAR) between 400 and 700 nm (perceived as white light) sometimes including the flanking regions of UV ~360–400 nm) and FR (~600–760 nm) radiation are emerging recently 0–400 nm ,38.−2 s−1 [For example, Spalholz et al. (2020) compared the response of two lettuce cultivars to a sun-simulated spectrum and other commonly applied B:R spectra, providing a biologically active radiation between 300–800 nm of 200 µmol m−2 s−1 . The stu−2 s−1 . In addi−2 s−1 . However−2 s−1 , and the−2 s−1 .Thus, it has been found that broad LED spectra, covering a wider plant-receptive spectral range rather than single narrow bands, and at best including flanking regions in the FR and UV, can lead to greater plant development. So far, however, such a broad LED spectrum has not been tested under insufficient light conditions in greenhouses. Therefore, our aim was to evaluate the advantages and disadvantages of broadband LED lighting during the winter season in northern central Europe  in a practical case study and to compare the results with the common HPS and FL setups found in the greenhouse industry and research facilities today.Thymus vulgaris L., which belongs to the Lamiaceae family rich in other genera such as Salvia and Organum [Thymus vulgaris L. is the monoterpene thymol [As a model plant, we chose moderately light-dependent  Organum  and whice thymol .The aim of this study was to conduct a greenhouse experiment during winter in order to assess the development, biomass, and health-promoting terpenoid yields of Thymus under a prototype broad-spectrum LED, as well as to obtain the prototypes’ power consumption and efficacy. To further evaluate the practical applicability and potential for greenhouse businesses and research facilities, we aimed at comparing the broad-spectrum LED results with results assessed under HPS and FL fixtures under their common setup conditions.Thymus vulgaris L. in comparison to the HPS and FL systems  in comparison to HPS (3.2 ± 0.5 g plant−1) and FL (0.6 ± 0.1 g plant−1), representing an increase of 1.75- and eight-fold, respectively (2 = 0.95 (p < 0.001), dry mass yields under the differing supplemental lighting systems were highly related to the individual daily light integrals (DLI).The LED system resulted in distinct yield increases, biomass partitioning, and a differentiated morphological appearance of  systems . While tunder FL A. Accordectively B. Therebectively C. With aThymus vulgaris L. was greatly increased under the LED system at the end of the experimental period and led to a profoundly different visual appearance in comparison to thyme plants grown under the other two supplemental lighting fixtures. Despite the lowest corresponding leaf-to-shoot ratio, which was 0.9 for LED, 1.3 for HPS, and 5 for FL, the leaf dry matter (LDM) of thyme was significantly increased and highest under LED (As indicated in nder LED C.2 = 0.95). That increasing DLIs accelerate the development and growth of plants up to a certain point is well established [The reason for the outstanding biomass accumulations and the concomitant rapid thyme development under the LED system is clearly found in the heightened DLI between 400 and 700 nm, as shown by the correlation coefficient of r = 0.97  only to approximately 7 mol m−2 d−1 during winter production, the FLs are neither suitable for the production of thyme nor presumably for the majority of greenhouse crops under the given cultivation conditions. HPS elevated the total DLI  to an estimated level of 11 mol m−2 d−1. Therewith, the biomass accumulation of Thymus vulgaris L. increased significantly in comparison to FL; however, the DLI remains insufficient for an optimal thyme production during winter. With a total DLI of approximately 16 mol m−2 d−1  during the low light season, the tested LED system achieved the highest DLI and approached the recommended DLI of ≥ 18 mol m−2 d−1 the most  in the leaf extracts of Thymus vulgaris L. under the supplemental lighting systems were thymol, γ-terpinene, and p-cymene, respectively, which is consistent with the results of former research [DM is highly enhanced by LED (2.7%) and HPS (2.3%) as compared to by FL (1.1%). The difference in quantity of VOCs per g of LDM between thyme plants cultivated under LED and HPS is not significant (p = 0.088). The LED considerably increased the amounts of all 13 evaluated terpenoids in the leaves of Thymus vulgaris L. in contrast to the FL system. The HPS system also enabled considerable increases in comparison to the FL system, even though the differences between the amounts of γ-terpinene and borneol are less profound, with p = 0.099 and p = 0.075, respectively. Differences between LED and HPS treatments were only detected for α-pinene, while myrcene (p = 0.077) as well as limonene (p = 0.057) differed only in tendency. All results are summarized in Applying GC-MS analysis, 12 monoterpenes and one sesquiterpene were identified in the leaf extracts of research ,49. TherZea mays L.) significantly increased their emissions of volatiles as light intensity increased up to 10,000 lm [Zea mays L. did not enhance any further, suggesting a kind of saturation or limitation was reached. The authors of [R/FR ratio [γ-terpinene, p-cymene and carvacrol, β-caryophyllene) and the number of essential oil-containing trichomes per cotyledone, two stimulatory effects that proved to be completely reversible by a subsequent exposure to far-red irradiation [Arabidopsis thaliana (L.) Heynh exposed to a low R/FR ratio of 0.2 as compared to plants exposed to a high R/FR ratio of 2.2 when controlling for light intensity [PFD 60 µmol m−2 s−1, PFD-R 7.6 µmol m−2 s−1) as well as their R/FR ratio (0.1) were significantly reduced as compared to LED and HPS under our experimental conditions. It would also suggest that the similar contents of VOCs per gram of thyme leaves found under LED and HPS are the result of their similar high R/FR ratios of 2.8 and 2.4, respectively. However, R/FR ratios dramatically decline under vegetational canopies. As described by Franklin (2008), a single leaf reduces a given R/FR ratio of 1.2 to 0.2 for the leaves growing underneath, and the ratio reduces further to 0.1 underneath a second leaf [Thymus vulgaris L. significantly increased under the LED system, it is reasonable to believe that the actual R/FR ratio underneath the more densely stands of thyme plants grown under the LED system was much lower than under the less dense canopy of thyme plants grown under HPS. This idea coincides with the result from Kegge et al. (2013), who detected a reduction of volatile emissions in plants grown in high density stands [Thymus vulgaris L. decrease with increasing proportions of blue light [R/FR ratios as well as under low R/B ratios may have been partially compensated by the LEDs’ elevated light intensity  compared to the intensity of the HPS system  in our study. Additionally, though air temperatures under the given experimental conditions did not differ between the supplemental lighting systems, it is known that leaf temperature increases under HPS lights as comparted to other lighting systems [Gouinguene and Turlings (2002) showed in their study that young corn plants , makingFR ratio . For exantensity . These fond leaf . As leafy stands . Anotherue light . The ass systems . As elev systems , a great systems .−2) with p < 0.06 . Both systems (LED and HPS) considerably promoted the VOC production in comparison to the FL system. DM and overall quantity of VOCs were highest for LED. Therefore, LED lightning offers an attractive alternative for thyme cultivation, both for essential oil production and delivery to fresh market.The broad-spectrum LED system enabled a highly significant increase in leaf and stem production of fresh thyme per square meter, representing increases of 43.3% and 82.4% in comparison to the HPS and FL system, respectively. Additionally, the dry matter productions of HPS and FL were highly reduced by 43.1% and 88.6% in comparison to the LED system. Further, the LED system enabled an increase in production of VOCs per square meter under the given greenhouse conditions in comparison to the conventionally used HPS system  was significantly higher than the dry thyme production under HPS (±155.2 mg) and FL (±39.1 mg). Further, the production of VOCs per kWh and square meter was significantly elevated underneath the LEDs (±5.4 mg) as compared to HPS (±1.9 mg) and FL (±0.4 mg). Results and calculations are combined in The LEDs consumed the least electricity with 257.7 W mOur power consumption results and thus the potential of LEDs for reducing energy costs coincide with numerous studies and reviews ,9,59, stFurther, in our study, plant growth may have been limited by nutrient availability, and an adjustment of fertilization based on the differing thyme growth rates may further increase biomass efficacies under HPS and especially under the broad-spectrum LED system. Nevertheless, when using the broad-spectrum LED lighting system, the significantly more inhomogeneous light intensity distribution compared to HPS and FL lamps  must be −2 nm−1, which are sufficient for high-quality thyme production. Therefore, if homogeneous plant development is not necessarily required, plants of marketable quality are also available with the LED setup used here without additional lamps.Nevertheless, at their edges, where the lowest light intensities occur, the LEDs achieve values of 16 W mThymus vulgaris L. grown under a broad-spectrum LED system, and to compare the lights’ productivity as well as electrical efficiency with conventionally used lighting fixtures for the cultivation of thyme under naturally low irradiated greenhouse conditions during fall and winter in Berlin, Germany, a one-factorial experiment with a randomized block design with three different supplemental light sources and four spatially independent replications  was conducted.To investigate biomass yields and contents of volatile organic compounds (VOCs) of ®, Berlin, Germany, dimensioning 47.5 × 21.5 × 19.5 cm3), (2) high-pressure sodium (HPS) lamps , and (3) fluorescent lamps (FL) ) were horizontally mounted onto given steel frames 1.40 m above greenhouse benches, resulting in distances between the bottom of the LED, HPS, and FL light sources and the greenhouse benches of 1.14, 1.13, and 1.09 m, respectively. Based on weather recordings from WetterKontor [Three different supplemental light sources ((1) fan-cooled light-emitting diode (LED)  and spectral irradiance (W m−2 nm−1) between 360 and 760 nm and photosynthetic photon flux density (PPFD in µmol m−2 s−1) between 400 and 700 nm. During a sunny day, light transmission between 350 and 800 nm of the natural irradiance through the greenhouse glass was determined to be 28% (±5%) by comparing the output of the spectrometer inside the greenhouse with the output outside the greenhouse and resulted in a photon flux density of ~434 µmol m−2 s−1 at bench level, which amounts to an approximate natural daily light integral of 3.9 mol m−2 d−1 when combined with the weather recordings  at night. Light intensity, spectral composition, and irradiance profiles (light distribution patterns) were measured and recorded at bench level under experimental conditions. The software package of the spectrometer (uSpectrum PC laboratory software) automatically calculated all electromagnetic parameters including photon flux density  were sown in 128-cell plug trays (Ø 4 cm) filled with potting substrate  on 9 October 2018 and placed under the differing lighting fixtures in a NS-orientated greenhouse located in Berlin, Germany . After six weeks on 20 November 2018, 32 representative seedlings were transplanted into pots (Ø 9 cm) containing substrate with an elevated nutrient composition  and evenly placed (quadratic) within 1 m2 under each light fixture. Each treatment was replicated four times, positioned in a randomized block design, and surrounded by 28 border plants to avoid boundary effects. Starting 27 December 2018, plants were fertilized weekly with 100 mL of a 0.2% (v/v) nutrient solution  containing 15% N, 10% P2O5, 15% K2O, 2% MgO, 0.01% B, 0.02% Cu, 0.075% Fe, 0.05% Mn, 0.001% Mo, and 0.015% Zn. However, due to the low biomass accumulation of the plants grown under the fluorescent lamp system, the plant fertilization was started two weeks later under the FL treatment. All thyme plants grew for a total period of 18 weeks until harvest on 12 February 2019. Climatic conditions including temperature and relative humidity of the greenhouse air were continuously monitored at canopy level via data loggers . Average temperatures (°C ± SD) under LED, HPS, and FL lighting were 20.4 ± 1.4, 21.1 ± 2.0, and 20.9 ± 1.2, with a measuring accuracy of 1 °C. Average humidities (%rh ± SD) under LED, HPS, and FL lighting were 47.2 ± 7.0, 44.2 ± 7.1, and 38.8 ± 7.1 with a measuring accuracy of 2.25%rh. Both climatic conditions did not differ between treatments.Seeds of DM) was determined for 16 plants selected from each treatment and replication, and the corresponding shoot dry matter (SDM) was calculated by subtracting the LDM from DM. All dried leaf samples were vacuum-sealed  and stored in the dark at 4 °C until further processing.To analyze the effect of the supplemental lighting systems on the yield, all 32 experimental thyme plants were harvested separately from each treatment condition and replication. Fresh matter (FM) of the above-ground plant parts was individually recorded at harvest on 12 February 2019. Total dry matter (DM) was measured after drying the samples in a circulated drying oven at 30 °C until stable mass was attained (≤seven days). Leaf dry matter  in order to estimate energy consumptions and biomass efficacies of the light fixtures. To correct for the detected difference between P and S due to heat dissipation of the HPS system, the measured cos phi of 0.93 was incorporated into the HPS’ power consumption calculations. According to the manufacturer’s specifications, the HPS system allows a homogeneously illuminated area of 1.56 m2 (1.2 × 1.3 m). Thus, the measured power consumptions were adjusted to the power consumption per square meter (W m−2) via rule of three. No adjustments were necessary for the LED and FL system.The power draw of current (I) and voltage (U) as well as electrical characteristics including real power (P) and apparent power (S) from representative lamps of each lighting treatment were measured using a power meter  were purchased as analytical standards with a purity of at least 95% for GC reference analysis from Alfa Aesar , Carl Roth , Merck KgaA , and Fluka . Isooctane  for solvent extraction of volatile organic compounds was obtained from Th. Geyer .Pure standard substances (n = 64) were extracted according to the following procedure: 100 mg (±2%) of gently oven-dried and powdered  thyme leaves were transferred into 2 mL screw cap micro tubes  including two steal grinding balls (Ø 2 mm). The plant material was homogenized in 1.0 mL of isooctane  6-methyl-5-penten-2-one as internal standard) for 10 min at 30 rps with a ball mill . After 10 min of ultra-sonication  and 10 min of centrifugation at 13,000 rpm  at 22 °C respectively, the supernatants were transferred into GC-vials and stored at −70 °C until analysis.The volatile organic compounds (VOCs) of 16 plants per treatment and replication , operating at 70 eV ionization energy, using the same temperature program as above. Helium was used as carrier gas with a constant flow rate of 1.2 mL min−1. Retention indices were calculated by using retention times of C6-C24-alkanes that were injected under the same chromatographic conditions.A total of 1 µL of the obtained extracts of volatiles was analyzed by GC–FID using an Agilent gas chromatograph 6890N fitted with an HP-5MS column (30 m × 250 µm × 0.5 μm) in splitless mode. Detector and injector temperatures were set to 250 °C. The following oven temperature program was used: 50 °C for 2 min, heating from 50 to 320 °C at a rate of 5 K minα-pinene, α- and γ-terpinene, β-caryophyllene, borneol, carvacrol, limonene, linalool, myrcene, p-cymene, sabinene, and thymol was confirmed by authentic reference standards by comparing their individual retention indices. Quantitative data of each main compound were obtained with serial dilutions of external standard solutions using at least six known concentrations , which covered concentration ranges detected for each compound in all samples.All main organic compounds of the volatile extracts were identified by comparing their mass spectra with those of internal reference libraries . Additionally, the identification of n = 4) were tested for normality with the Shapiro–Wilk test, and all data sets passed the normality test at α = 0.05. Finally, all data sets were statistically analyzed using a Brown-Forsythe and Welch ANOVA test due to unequal variances between treatments. Multiple comparisons were conducted via Dunnett T3. Two-tailed Pearson correlation between biomasses and DLIs was calculated after first computing means of 18 replicates per spatially independent light treatment and then analyzing those means (n = 12). Biomass efficacies (g or mg kWh−1 m2) were calculated based on electricity consumed within a square meter during the cultivation period.Statistical analysis was performed using GraphPad Prism 8.4.2.679 . Data of each spatial replication were tested for normality via Anderson-Darling, D’Agostino and Pearson, Shapiro–Wilk test, and Kolmogorov–Smirnov test. If normality test failed, outliers were identified via ROUT method (Q = 10%) and removed to establish normality of all data sets. The means of each spatial replication (Thymus vulgaris L. can be achieved faster and thus more often when replacing HPS and FL light sources with the tested LED system, ultimately resulting in greater revenues at simultaneously highly reduced production costs for greenhouse growers. The comparatively high initial capital costs of LEDs which have delayed their establishment in the past are decreasing [With its outstanding biomass as well as terpenoid efficacy, the broad-spectrum LED system represents a strong competitor to the conventionally used HPS and FL lighting systems in greenhouses under naturally insufficient light conditions as investigated in this study. Marketable creasing . Based ocreasing ,15,63,64creasing ,65 can fcreasing ,66. Our


Para-sulfocalix[n]arenes are promising host molecules that can accommodate various chemotherapeutic drugs. Pt(IV)-based complexes, including satraplatin and asplatin, are promising alternatives that overcome the shortcomings of Pt(II) complexes. In this study, asplatin has been synthesized by fusing acetylsalicylic acid (aspirin) and cisplatin. Furthermore, it has been characterized using 1H NMR, mass spectrometry, elemental analysis, and UHPLC. A host-guest complex of asplatin and p-sulfocalix[4 M–1 and 8.7 × 104 M−1, which corresponds to a free energy of complexation of −6.8 kcal mol–1, while the calculated value for the inclusion free energy is −13.2 kcal mol−1. Both experimentally and theoretically estimated complexation free energy show that a stable host-guest complex can be formed in solution. The in vitro drug release study displayed the ability of the complex to release its cargo at a cancerous pH (pH of 5.5). Additionally, the asplatin/PSC4 complex is shown to be biocompatible when tested on human skin fibroblast noncancerous cells, demonstrating the highest in vitro cytotoxic activity against (MCF-7), cervical (HeLa), and lung cancer cells (A-549), with IC50 values of 0.75, 2.15, and 3.60 µg/mL, respectively. This is as compared to either cisplatin  or asplatin . On the other hand, the free asplatin exhibited higher cytotoxicity on cancerous cells and lower toxicity on noncancerous cells. The outcomes of the present joint theoretical and experimental investigation reinforce the interest in platinum-based anticancer therapeutics when they are protected from undesired interactions and suggest the use of the PSC4 macromolecule as a promising carrier for Pt(IV) anticancer drugs. The formed asplatin/PSC4 inclusion complex may represent an effective chemotherapeutic agent.ulfocalixarene (PS Tests have proved asplatin to exhibit more potent anticancer activity and lower adverse effects when compared to cisplatin arenes [Para-sulfonatocalix[n]arenes (PSC4) are water-soluble (>0.1 mol L−1) and innocuous to human cells at doses up to 109 ng/kg [para-sulfonatocalix[n]arenes can accommodate numerous guest molecules in their cavities via the host-guest inclusion complexation arenes (CXs), cyclodextrins (CDs), cucurbiturils (CBs), and pillararenes, have been suggested as smart drug delivery platforms ,22,23 anargeting ,25,26. Cn]arenes . Para-suluble >0. mol L−1 1H NMR spectroscopy and mass spectrometry. Furthermore, a novel UHPLC method has been developed for detecting asplatin. Then, complexation between Para-sulfonatocalix[n]arenes (PSC4) and asplatin in an aqueous medium has been investigated for possible application in cancer therapy. The developed complex has been characterized by UV, UHPLC, and computational studies. The stoichiometry and binding constants have been determined by Job’s plot (continuous variation method) and quantum mechanical calculations at the density functional theory (DFT) level. Theoretically, the structural characterization of the formed host-guest complexes has been carried out. Intermolecular hydrogen bonds in the asplatin/PSC4 adducts have been studied by means of the atoms in molecules (AIM) theory. Moreover, the cytotoxic activities of the complex against breast adenocarcinoma (MCF-7), cervical cancer (HeLa), and lung cancer (A549) have been studied and compared to free asplatin and cisplatin. The biocompatibility of the designed complex has also been evaluated using human skin fibroblast noncancerous cells and compared to free asplatin and cisplatin.In this work, asplatin has been synthesized and characterized using Asplatin has been synthesized as reported previously with some modifications ,28.The synthetic pathways of acetylsalicylic acid anhydride and asplatin are presented in 1H-NMR spectroscopy , as presented in 2O-exchangeable signals of a hydroxyl and two amine groups, respectively.The formation of asplatin has been confirmed using The melting point for asplatin was 139–141 °C. Furthermore, mass spectrometry and elemental analysis were used to confirm the synthesis of asplatin .1H-NMR was used to confirm the formation of a 1:1 M ratio asplatin/PSC4 complex  may suggest hydrogen bonding within the host.Furthermore,  complex . The aroAnalysis of the synthesized asplatin using UHPLC showed a sharp symmetric peak at a retention time of 0.736 min A. MeasurThe host-guest inclusion complexation between asplatin and PSC4 in an aqueous medium has been studied applying UV-Vis spectroscopy .The UV absorbance spectra of mixtures of 0.2 mM PSC4 and increasing asplatin concentrations (ranging from 0.01–0.25 mM) have been recorded in aqueous media . The spe−1, corresponding to a complexation free energy of −6.8 kcal mol−1. This value is within the stability constant range (0.001 × 104 to 16 × 104 M–1) of host-guest complexes designed for drug delivery applications arene, PSC4, was purchased from BLD Pharmatech Company . Streptomycin, penicillin, fetal bovine serum, trichloroacetic acid (TCA), Dulbecco’s Modified Eagle’s Medium (DMEM) SRB, and tris(hydroxymethyl)aminomethane (TRIS) were purchased from Lonza, Basel, Switzerland. All other reagents have been obtained from Sigma Aldrich .1H-NMR analysis was carried out using a JEOL type GSX-270 or JNM-ECA 500II spectrometer . Mass spectrometry was conducted on a Shimadzu GCMS-QP 5050A gas chromatograph-mass spectrometer. HPLC measurements were carried out using an Agilent ultra-performance 1290 infinity, equipped with 1290 DAD, a gradient quaternary pump VL, an auto-sampler ALS, a column oven TCC and a 1290 Thermostat. Data acquisition was performed using Agilent Chemstation software (B.04.03), and data processing was subsequently performed using Agilent LabAdvisor (Utility) Quantitative analysis software (B.02.04). UV spectrophotometric measurements were carried out on a CARY 500 UV–Vis-NIR Scan dual-beam spectrophotometer .The The synthesis of asplatin was conducted as reported elsewhere, with some modifications ,28. Firs2O2  added dropwise. The reaction was allowed to take place for 1 h at an increasing temperature from 50 °C to 100 °C; then, it was stirred for an extra 12 h at room temperature. The resultant was lyophilized, washed with cold water and ethanol and dried in a vacuum. Finally, asplatin was prepared by reacting a solution of oxoplatin  in 15 mL dimethylsulfoxide (DMSO) with the previously prepared acetylsalicylic acid anhydride , and the mixture was stirred for 24 h at room temperature. The obtained residue was washed with acetone and ether and dried under a vacuum. The final product of asplatin was collected as dark yellow crystals of yield 78% and characterized by different techniques.Furthermore, oxoplatin was prepared via reacting 374 mg of cisplatin (1.23 mmol) suspended in 15 mL distilled water with 12.5 mL of H1H-NMR  and showed the following signals: δ 7.77–7.76 , 7.47–7.44 , 6.90–6.89 , 6.87–6.86 , 2.98 , 2.52 , 2.49  and 2.33 .The structural elucidation of asplatin was done by The asplatin/PSC4 complex was prepared by mixing 0.2 mM PSC4 with different concentrations of asplatin (ranging from 0.01–0.25 mM) in aqueous media.1H-NMR showed the following signals: δ 12.48 , 7.75–7.73 , 7.41–7.40 , 6.87–6.85 , 6.83–6.82 , 6.0–5.80 , 3.34 , 2.52  and 2.49 .The structural elucidation of the asplatin/PSC4 complex by The complex formation and stoichiometry were studied using UV-Vis Spectroscopy and UHPLC.v/v). The flow rate was 0.2 mL/min at a 30 °C column temperature with an injection volume of 1 µL.The synthesized asplatin and its complex with PSC4 were analyzed using a UHPLC diode array and a Zobrax Eclipse Plus C18 column . The mobile phase consisted of acetonitrile: 0.1% orthophosphoric acid in the ratio of . The dialysis bag was placed into 25 mL of PBS at pH 7.4 or 5.5. The whole system was placed in a shaking incubator  and rotated at 100 rpm with a temperature maintained at 37 °C. At definite time intervals, a 1 mL aliquot of the sample was withdrawn for analysis by UHPLC and immediately replaced with another equal volume of warm buffer .v/v) CO2 at 37 °C.Breast adenocarcinoma cells (MCF-7), cervical cancer cells (HeLa), lung cancer cells (A-549), and human skin fibroblast healthy cells were obtained from American Type Culture Collection  and sustained in DMEM medium complemented with streptomycin (100 mg/mL), penicillin (100 units/mL), and 10% heat-inactivated fetal bovine serum. Cells were incubated in 5% (50 (in μg/mL) value was detected employing our previously reported methods [3 cells) was seeded in 96-well plates and incubated in DMEM medium for 24 h at 37 °C and 5% CO2. Cells were treated with medium (100 μL) containing different concentrations (from 0.01 to 300 μg/mL) of PSC4, cisplatin, asplatin, and asplatin/PSC4 complex. After 48 h, culture media were detached, and 10% TCA (150 μL) was added at 4 °C for 1 h. The cells were then washed several times with phosphate buffer saline. Then, 70 μL of SRB solution (0.4% w/v) was added and incubated in the dark for 10 min at room temperature. Acetic acid (1%) was used to wash the plates, and then the plates were left to dry for 24 h. Afterward, the protein-bound SRB stain was liquefied using 10 mM TRIS (150 μL), and absorbance was measured at 540 nm  [Breast adenocarcinoma cells (MCF-7), cervical cancer cells (HeLa), lung cancer cells (A-549), and human skin fibroblast healthy cells were treated with various concentrations of PSC4, cisplatin (positive control), asplatin, and asplatin/PSC4 complex. The in vitro cytotoxic activities against either cancerous or noncancerous cells were evaluated utilizing Sulforhodamine B Colorimetric Assay (SRB assay), and the IC methods ,17,18. CGermany) ,17,18.All calculations were carried out at the DFT level of theory using the Gaussian16 package . For theThe solvation effects were computed using the continuum solvation model based on density (SMD),  as impleFrequency calculations within the harmonic approximation were also carried out at the same level of theory to explore and confirm the nature of the intercepted stationary points as minima and to calculate the Gibbs free energies for the inclusion of asplatin (G) into calix-4 arene (H).The inclusion Gibbs free energy, in solution The basis set superposition error (BSSE) estimated using the Boys–Bernardi counterpoise technique  was evalThe quantum theory of atoms in molecules (AIM) suggested by Bader  was usedω and the signature σ mark the CPs. The rank of the diagonalized matrix is given by the number of non-zero eigenvalues, namely non-zero curvatures of ρ(r) at the CPs. The signature of a CP, σ, is defined as the algebraic sum of the signs of eigenvalues (signs of curvatures of ρ(r) at the CPs). A CP with ω = 3 and σ = +1, corresponding to  CP, has 2 positive curvatures, and ρ is a minimum at the CP in the plane defined by their corresponding axes. In contrast, at the CP along the third axis perpendicular to this plane, ρ is a maximum. The CP , with ω = 3 and σ = −1, highlights that electronic charge density is located within a chemical bond between the involved atoms. Such a point is named a bond critical point (BCP).The analysis of the electron density –1 and −13.2 kcal mol–1, respectively. Asplatin/PSC4 displayed stronger anticancer activities against MCF-7 as compared to oxaliplatin/PSC4 and carboplatin/PSC4 complexes. Additionally, the asplatin/PSC4 complex showed a more potent anticancer activity than the carboplatin/PSC4 complex against the A-549 cancer cell line. These findings demonstrate the promising potential of the asplatin/PSC4 complex as a biocompatible chemotherapeutic agent and also support the use of PSC4 as a delivery system for metal-containing chemotherapeutics and as a scaffold for designing novel carriers for antitumor prodrugs.The synthesis and the characterization of the asplatin prodrug and the study of its encapsulation into PSC4 to form a supramolecular adduct have been reported. The data reported in this work shows that the Pt(IV) complex can generate a supramolecular system with the PSC4 cavity, improving its cytotoxicity and biocompatibility profiles compared to free asplatin. The structural features of the system are well supported by the method of continuous variation (Job’s plot) and DFT analysis. The host-guest complex that involves the insertion of asplatin within the cavity of PSC4 is mainly stabilized by forming hydrogen bonds between the axial ligands of the platinum(IV) prodrug and a sulfonate moiety of the host cavity. The two most stable inclusion arrangements have been selected, and the corresponding estimated experimental and theoretical values of the free energy of complexation in solution are −6.8 kcal mol


Gls), ionotropic and metabotropic glutamate receptors , and glutamate transporters (Slc17a7). SANPCs highly expressed cell markers of glutamatergic neurons (Snap25 and Slc17a7), whereas Gad1, a marker of GABAergic neurons, was negative. Functional studies revealed that inhibition of glutamate receptors or transporters reduced spontaneous pacing frequency of isolated SAN tissues and spontaneous Ca2+ transients frequency in single SANPC. Collectively, our work suggests that SANPCs share dominant biological properties with glutamatergic neurons, and the glutamatergic neurotransmitter system may act as an intrinsic regulation module of heart rhythm, which provides a potential intervention target for pacemaker cell-associated arrhythmias.Activation of the heart normally begins in the sinoatrial node (SAN). Electrical impulses spontaneously released by SAN pacemaker cells (SANPCs) trigger the contraction of the heart. However, the cellular nature of SANPCs remains controversial. Here, we report that SANPCs exhibit glutamatergic neuron-like properties. By comparing the single-cell transcriptome of SANPCs with that of cells from primary visual cortex in mouse, we found that SANPCs co-clustered with cortical neurons. Tissue and cellular imaging confirmed that SANPCs contained key elements of glutamatergic neurotransmitter system, expressing genes encoding glutamate synthesis pathway (The online version of this article (10.1007/s13238-020-00820-9) contains supplementary material, which is available to authorized users. SANPCs expressed not only cell markers of glutamatergic neurons, but also key components of the glutamatergic neurotransmitter system. Functional studies revealed that antagonists of glutamate receptors or transporters dramatically decreased the spontaneous pacing frequency of isolated SAN tissues and the frequency of spontaneous CaHcn4 . Of these, Clusters 1 and 2 each contained three transcriptomic cell subpopulations . Based on the expression of canonical markers in the nervous system, we observed that the entire cell population could be segregated into distinct cell types, corresponding to glutamatergic neurons (Slc17a7+), GABAergic neurons (Gad1+), oligodendrocyte precursor cells (Pdgfra+), microglia (Itgam+), astrocytes (Aqp4+) and endothelial cells (Flt1+) .To provide an overall view of SANPCs and CCs, we profiled the single-cell transcriptome analysis of 718 SANPCs from 21 adult mice. With Seurat, we compared these SANPCs with 1,809 CCs, which contained diverse cell types, including glutamatergic neurons, GABAergic neurons and non-neuronal cells  enrichment analysis of differentially expressed genes (DEGs) indicated that the DEGs enriched in each cell cluster corresponded to their characteristic biological processes. For example, Cluster 1 was associated with intracellular signal transduction and neurotransmitter transport. Cluster 2 expressed genes involved in cell-cell signaling and chemical synaptic transmission , ionotropic and metabotropic glutamate receptors , and glutamate transporters (Slc17a7)  were lowly expressed in SANPCs  Fig. A. The exSnap25), vesicle-bound glutamate transporter VGLUT1 (Slc17a7), and glutamate decarboxylase GAD1 (Gad1) in SAN sections and single SANPC by immunofluorescence. Our results confirmed the expression of SNAP25 and VGLUT1 in SANPCs, while the expression of GAD1 was not observed  rather than GABAergic neurons .We also detected the protein expression of cell marker genes for both glutamatergic neurons and GABAergic neurons, including presynaptic plasma membrane protein SNAP25  were also highly expressed in SANPCs , acting as glutamate-gated ion channels, and metabotropic glutamate receptors (mGluRs), acting via G proteins. iGluRs are classified into three major subtypes: N-methyl-d-aspartate receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) and kainate receptors  were 97.23 μmol/L : 65.51–157.6), (-)-MK801) and 668.3 μmol/L  -MK801, a non-competitive NMDAR antagonist, and MTEP hydrochloride, a non-competitive mGluR5 receptor (a subtype of mGluRs) antagonist, caused a concentration-dependent decrease in spontaneous pacing frequency of SAN tissues Fig. A–C. The 50 = 58.52 μmol/L, 95% CI: 37.51 to 91.05)  are important in the removal and uptake of glutamate from the extracellular space, playing a critical role in the regulation of glutamatergic neurotransmission in the central nervous system , 37.38 ± 15.33 bpm (MTEP hydrochloride) and 58.4 ± 36.39 bpm , respectively , while the data of 1,809 mouse cortical cells were obtained from other research group (GSE71585). Seurat clustering analysis was used to determine the transcriptomic relationship between individual cells, while the neuronal features of SANPCs and CCs were defined by the gene expression pattern of synthesis, receptors and transporters of neurotransmitters.This study conformed to the rules of the Guide for the Care and Use of Laboratory Animals made by the U.S. National Institutes of Health. All the animal experiments were approved by the Animal Care and Use Committee of Tongji University School of Medicine.2PO4, 1.8 mmol/L CaCl2, 1.0 mmol/L MgCl2, 5.5 mmol/L glucose and 5 mmol/L HEPES (pH was adjusted to 7.4 with NaOH). The solution was continuously oxygenated with O2. The SAN was dissected from the region bordered by the crista terminalis and the superior and inferior vena cava. The SAN tissue was cut into small pieces and washed twice in low-Ca2+ solution containing: 140 mmol/L NaCl, 5.4 mmol/L KCl, 1.2 mmol/L KH2PO4, 0.2 mmol/L CaCl2, 18.5 mmol/L glucose, 50 mmol/L taurine and 1.0% BSA (pH was adjusted to 6.9 with NaOH). The pieces of SAN tissue were then digested at 36–37 °C for 20–25 min in low-Ca2+ solution containing elastase , collagenase type II  and protease  with gentle agitation. The digestion was quenched by transferring the pieces to modified Kraftbruhe solution, containing: 100 mmol/L potassium glutamate, 10 mmol/L potassium aspartate, 25 mmol/L KCl, 10 mmol/L KH2PO4, 2 mmol/L MgSO4, 20 mmol/L taurine, 5 mmol/L creatine, 0.5 mmol/L EGTA, 20 mmol/L glucose, 5 mmol/L HEPES and 1.0% BSA (pH was adjusted to 7.2 with KOH).SANPCs were isolated from male C57BL/6 mice (8–12 weeks of age) as previously described  and then amplificated using KAPA polymerase , The product was purified using Ampure XP Beads  for Nextera Tagmentation , then the libraries were sequenced with paired end 2 × 150 bp reads on Illumina HiSeq X10 to generated the raw sequencing data.Raw data of scRNA-seq of SANPCs were initially trimmed with Trimmomatic (V0.33). Then the obtained clean reads were aligned to mouse reference genome (GRCm38), using Hisat2 (V2.0.5), and annotated using GENCODE (release 20). Finally, the gene expression was quantified by read counts.Read count of SANPCs and CCs were used as input to Seurat V2 R package (V2.3.4) for the clustering analysis. The Seurat objects of SANPCs data and CCs data were generated using “CreateSeuratObject” function. Then “FilterCells” function was used for dropping the low-quality cells out which expressed less than 1000 or more than 12,500 genes. After the data were normalized and scaled by “NormalizeData” and “ScaleData” function, 2,000 high variable genes were calculated separately in SANPCs and CCs datasets and then merged into 3,290 unique high variable genes across two datasets. Subsequently, the batch effects of these two objects were removed and then combined into an integrated object using “RunCCA” and “AlignSubspace” function, basing on the canonical correlation analysis alignment in Seurat. 15 CC components were chosen for the dimensionality reduction according to the result of “MetageneBicorPlot” function, which examined a measure of correlation strength for each CC . Then we performed tSNE analysis through “RunTSNE” and “FindClusters” function with 0.6 resolution. Diverse cell subpopulations were identified and annotated using the canonical neuronal cell markers.We also used Seurat V3 R package (V3.2.2) for the batch effect correction between SANPCs data and CCs data with similar parameters. 2,000 variable genes were identified using “FindVariableFeatures” function for the PCA analysis. “FindIntegrationAnchors” function was used to integrate the two objects. Based on the results of “ElbowPlot” and “JackStraw” functions, 15 PCs were chosen for the dimensionality reduction, then the cell clusters were identified using the “FindNeighbors” and “FindClusters” functions.In addition, Seurat V3 (V3.2.2) was also used for the integration analysis between CCs and embryonic SANPCs, the scRNA-seq data of which was obtained from SAN and adjacent atrial tissues of E13.5 embryos (GSE130461)  were quickly removed, preserving the atrium and peripheral connections. After rinsing with cold PBS, the hearts were fixed with 4% paraformaldehyde  overnight at 4 °C. Then, the hearts were dehydrated in gradually increased concentrations of ethanol and then embedded in paraffin. Hearts were sectioned horizontally in 6 μm sections. The SAN of adult mice located in the posterior wall of the right atrium, in the intercaval region adjacent to the atrial muscle of the crista terminalis, extending from the superior to near the inferior vena cava. The SAN is surrounded by connective tissue, and the SAN artery passing through the area.After deparaffinization, re-hydration and microwaving for antigen retrieval in citrate solution, paraffin-embedded heart slices were blocked with 10% goat serum  and incubated with primary antibody overnight at 4 °C. The following day, the slices were washed three times in PBST and then incubated with respective fluorescent secondary antibody  for 1h at room temperature. The slices were washed again in PBST and then stained with DAPI  to label the nuclei. Pictures were taken from a confocal microscope .Isolated SANPCs were attached to glass coverslips precoated with laminin. For immunofluorescence, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature. After fixation, the SANPCs were permeabilized with 0.5% Triton X-100 for 10 min and then blocked with 4% normal goat serum in PBS for 1 h at room temperature. Next, the cells were incubated with primary antibodies overnight at 4 °C and washed with PBS (3 times for 5 min each). Then, the cells were incubated with Alexa Fluor conjugated second antibodies for 1h at room temperature. The nuclei were stained with 1 μg/mL DAPI for 5 min. The negative control of immunostaining in SANPCs was performed with secondary antibody incubation only . The cells were imaged by confocal microscopy  and analyzed using LAS X software.2PO4, 1.8 mmol/L CaCl2, 1.0 mmol/L MgCl2, 5.5 mmol/L glucose and 5 mmol/L HEPES . The field potential recordings were acquired as previously described  were dissected from the region bordered by the crista terminalis and the superior and inferior vena cava. The SAN tissues were placed in a warm tissue bath with oxygenated Tyrode’s solution: 140 mmol/L NaCl, 5.4 mmol/L KCl, 1.2 mmol/L KH2+ release of SANPCs was measured by IonOptix Imaging System  and analyzed using the IonWizard™ v6.1 acquisition software.The isolated SANPCs were incubated in Tyrode’s solution were loaded with 0.5 μmol/L fluo-4 AM  for 5–10 min at room temperature. After centrifuged, the supernatant was removed, and the pellet was re-suspended in the Tyrode’s solution. Then, the cells were placed in a heated chamber, mounted on a Leica DMI3000B inverted microscope, and visualized at 100× magnification. Fluorescent images were collected alternately at excitation wavelengths of 488nm with an emission wavelength range from 500 nm to 550 nm. The CaAntibodies included those against GLS , GRINA , GLUR3 , mGluR1 , mGluR5 , VGLUT1 , UNC13B , SNAP25 , GAD1 , SYT11 , CAST , and HCN4 . Alexa Fluor conjugated secondary antibodies were purchased from Abcam.t-test (two sided) and mixed-effects model in each of the specific experimental designs presented in the figures. P < 0.05 was considered as statistically significant.Statistical analyses were performed with GraphPad Prism 8.0 software. The data are presented as mean ± SEM. No statistical methods were used to predetermine sample size. Normality was tested with the Kolmogorov-Smirnov test. Groups were compared using an unpaired Student’s Supplementary material 1 (PDF 1166 kb)Below is the link to the electronic supplementary material.


Cellulose acetate (CA), polyimide (PI), cellulose triacetate (CTA), polyethersulfone (PES) and polysulfone (PSU) membranes were fine-tuned as materials for preparation of NF-membranes, either by selecting a suitable non-solvent for phase inversion or by increasing the polymer concentration in the casting solution. The best membranes were prepared with CTA in GVL using water as non-solvent: with increasing CTA concentration (10 wt% to 17.5 wt%) in the casting solution, permeance decreased from 15.9 to 5.5 L/m2·h·bar while RB rejection remained higher than 94%. The polymer solubilities in GVL were rationalized using Hansen solubility parameters, while membrane performances and morphologies were linked to viscosity measurements and cloudpoint determination of the casting solutions to better understand the kinetic and thermodynamic aspects of the phase inversion process.γ-Valerolactone (GVL) was selected as a renewable green solvent to prepare membranes via the process of phase inversion. Water and ethanol were screened as sustainable non-solvents to prepare membranes for nanofiltration (NF). Scanning electron microscopy was applied to check the membrane morphology, while aqueous rose Bengal (RB) and magnesium sulphate (MgSO Membrane technology offers separations in the chemical industry and water treatment of small molecules (solutes) from solvent or water streams by using membranes and providing an economically viable alternative for separation and purification ,2. In naSeveral techniques have been applied to prepare polymeric membranes, including temperature and non-solvent induced phase separation (TIPS and NIPS) ,4,5. AmoAs membrane processes are appearing increasingly in industrial applications, it is becoming more important that membrane preparation itself becomes “green” ,20,21,22According to principles 5 and 7 of the 12 principles of green chemistry, safer solvents and auxiliaries and use of renewable feed stock are main aspects of green chemistry ,28. Not olactone  have bee(207 °C) ,38 and h(207 °C) ,38,39. G(207 °C) .A related lactone-based solvent, and TIPS ,41. In tPolysulfone  and polyimide (Matrimid) and were provided by Solvay (Belgium) and cellulose triacetate by Eastman (Belgium). Cellulose acetate, polyethersulfone and GVL were purchased from Sigma-Aldrich (Belgium). All polymers were dried for 24 h at 105 °C. Molecular weight (MW) and the structure of the polymers, feed solutes and solvent used in this work are given in All polymers were dissolved in GVL at room temperature and stirred magnetically over 24 h by dissolving 10–20 wt% of the polymer (except for cellulose triacetate (CTA), where 17.5 wt% was found to be the upper concentration limit) in GVL, for details see The viscosity or rheological measurements for all polymer samples were done on a Anton Paar MCR 501 (Austria) with cone-plate geometries and evaporation blocker, as described in literature ,10,42.The procedure for cloudpoint determination was adapted from literature, as described elsewhere ,10,42 us2. Solutions of 35 µM rose Bengal (RB) or 16.7 mM MgSO4 in distilled water (H2O) were taken as feed -spectrophotometer at a wavelength of 548 nm. In the case of MgSO4, a Consort K620 conductometer was used to measure the concentration of permeate and feed.Filtrations were performed at 23 °C under pressures from 2 to 16 bar permitting filtrations of 16 membrane coupons simultaneous with a high-throughput filtration set-up ,44. Each as feed . PermeanFor membrane morphology studies, membrane samples were broken in liquid nitrogen and coated with 2–5 nm gold/palladium as conductive layer using a Jeol-AFC HR-sputter coater. Images were acquired using a JEOL JSM 6010LV scanning electron microscopy (SEM).Ra (solubility parameter distance) was calculated using Equation (3), which is a measure for affinity between polymer (1) and solvent (2).The Hansen solubility parameters (HSP) are used to describe the affinity between a polymer and GVL, as discussed earlier ,42,45,46Values for GVL are taken from literature ,46,47,48To understand the role of polymer concentration in NIPS, both kinetic and thermodynamic aspects are studied in detail to see the effect on final membrane morphology and performance. Kinetics play a role in phase inversion via the diffusion of non-solvent into the polymer solution and of solvent out of the cast polymer film. These diffusion rates obviously depend on the molecular size and the viscosity of the polymer solution. Depending on this solvent/non-solvent exchange rate and the strength of the non-solvent  between solvent/non-solvent and between non-solvent/polymer were calculated  measurements of the casting solutions. The viscosities of the polymer solutions logically increased with increasing polymer concentrations . Polymer2·h·bar was found for CA10W which logically decreased to 0.70 and 0.30 L/m2·h·bar respectively with RB rejection increasing from 49% to 95% for CA15W and CA20W membranes  , were also tested using a MgSO2·h·bar) with all RB rejections above 90%. When selectivity is more important, PES membranes are preferred with 68% MgSO4 and a permeance of 1.1 L/m2·h·bar. Permeances and MgSO4 rejections are in general low as compared to commercial membranes which typically range from 1.0 to 16.3 L/m2·h·bar and 60.0% to 99.2% rejections [4 rejections were lower.When aiming for a high-permeance, CTA membranes  are the best option  with sustainable solvents are always suggested over petroleum-based ones. However, there is currently a tradeoff still between selectivity and sustainability.To combine high rejection with a high permeance seems challenging for polymer/GVL systems within the studied parameters space of polymer type, polymer concentration and NS choice. However, other parameters like, e.g., membrane annealing, coagulation bath temperature and composition, co-solvent addition in the casting solution can still be screened to further optimize these properties. γ-butyrolactone (GBL) was previously used in membranes preparation, e.g., polyvinylidene flouride (PVDF) membranes were prepared via TIPS and polyetheretherketone (PEEK-WC) membranes via NIPS. However, the use of GBL was limited to these 2 polymers. While GVL not only replaced GBL, but it also provided more opportunities to prepare NF membranes as it can be combined with common membrane polymers, like CA, PI, PES, CTA and PSU, making it an interesting solvent for membrane preparation, superior to GBL.NF membranes based on CA, PI, CTA, PES and PSU have been successfully prepared using GVL as a bio-based green solvent via NIPS.2·h·bar and a 94% RB-rejection. Other membranes prepared from PES, PI, PSU and CA had a reasonable permeance with RB-rejection over 90%.For all polymer types, the NF-criterion with RB-rejections above 90% could be fulfilled by tuning the membrane preparation using water or ethanol as non-solvent. The best membrane, CTA10W, was prepared using water as a non-solvent, from low CTA concentration. It had a permeance of 15.9 L/mLoose NF membranes prepared by using polymer/GVL systems still have potential to be further tuned toward tight NF, but can obviously already serve as a ultrafiltration membrane or as a support layer in thin film composite preparation.


E. coli solution. Results showed that DHA treatment concentration-dependently promoted the accumulation of BMMSCs in prostate tissue of CBP mice. In addition, DHA and BMMSCs cotreatment significantly alleviated inflammation and improved prostate damage by decreasing the expression of proinflammatory factors such as TNF-α, IL-1β, and chemokines CXCL2, CXCL9, CXCL10, and CXCL11 in prostate tissue of CBP mice. Moreover, DHA and BMMSCs cotreatment displayed antioxidation property by increasing the production of glutathione peroxidase (GSH-Px), SOD, and decreasing malondialdehyde (MDA) expression. Mechanically, DHA and BMMSCs cotreatment significantly inhibited the expression of TGFβ-RI, TGFβ-RII, phosphor (p)-Smad2/3, and Smad4 in a dose-dependent manner while stimulated Smad7 expression in the same manner. In conclusion, our findings provided evidence that DHA effectively eliminated inflammatory and oxidative stress against prostate injury, and this effect involved the TGF-β/Smad signaling pathway in CBP.Although bone marrow mesenchymal stem cells (BMMSCs) are effective in treating chronic bacterial prostatitis (CBP), the homing of BMMSCs seems to require ultrasound induction. Dihydroartemisinin (DHA) is an important derivative of artemisinin (ART) and has been previously reported to alleviate inflammation and autoimmune diseases. But the effect of DHA on chronic prostatitis (CP) is still unclear. This study aims to clarify the efficacy and mechanism of DHA in the treatment of CBP and its effect on the accumulation of BMMSCs. The experimental CBP was produced in C57BL/6 male mice via intraurethrally administered  The clinical manifestations of CP include abnormal urination, pain, and sexual dysfunction, especially symptoms of voiding dysfunction . Based oherapies . Howeverherapies . Many TCnangiana , Bazhengnangiana , and flanangiana  were eff15H24O5) is a sesquiterpenoid compound and is an important derivative of ART. As DHA research progresses, the pharmacological benefits of DHA were not only limited to the treatment of malaria but also showed great promise in anti-inflammatory and immune regulation. Previous studies have found that DHA was effective in the treatment of inflammatory diseases and oxidative stress, especially for acute kidney injury [Artemisinin (ART) was a well-known clinically important antimalarial drug, which was extracted from Artemisia annual leaves and was first discovered and reported by Professor Tu Youyou, an outstanding pharmacologist in China . Dihydroy injury , pulmonay injury , and airy injury . It is py injury . Furthery injury , antitumy injury , 14, andy injury , 17. They injury . Howeverα and IL-1β expressions [Bone marrow mesenchymal stem cells (BMMSCs) are a stem cell population with rapid population proliferation, multidirectional differentiation, low immunogenicity, immune suppression, and tissue repair ability . BMMSCs ressions . In thisDHA was purchased from KPC Pharmaceuticals, Inc. . Product batch number: C00220160402.C57BL/6 male mice  were purchased from Dashuo Animal Experiment Co., Ltd.  2020–034). The feeding environment was 25 ± 1 °C, relative humidity 50%–60%, and light/darkness for 12-h circulation. The mice are allowed to freely eat and drink.n = 6), namely, the control group, model group, BMMSCs group, low-dose DHA + BMMSCs group, and high-dose DHA + BMMSCs group. All mice were anesthetized with 1% sodium pentobarbital  prior to treatment. For the control group, the male mice were instilled with 200 μl phosphate buffer saline (PBS); for the model group, the mice were intraurethrally administered with 200 μl of E. coli solution (2 × 106 cfu/ml). After modeling, all mice were normally fed for 4 weeks. Then, for the BMMSC group, the mice were injected with 30 μl of PBS buffer containing 1 × 107 GFP-BMMSCs in the tail vein; for DHA low- and high-dose groups, the mice were given DHA 10 mg/kg/d and 20 mg/kg/d by gavage, respectively. The control and model groups were given 0.9 g/L saline 3 ml/d by gavage. All mice were raised in an SPF environment for another 2 weeks. Finally, the mice were anesthetized with 1% sodium pentobarbital (50 mg/kg) and then euthanized. The prostate tissue was removed for subsequent analysis.The experimental protocol for the care and use of laboratory animals was approved by the Experimental Animal Ethics Committee of Shijiazhuang City People's Hospital (2021097). The mice were randomly divided into 5 groups  were seeded in a 96-well plate and cultured in an incubator  for 24 h. Subsequently, 100 μl diluent of GFP-LUC adenovirus (1 × 109 cfu/ml) was transfected into BMMSCs and cultured for 24 h. A fluorescence microscope was used to observe the expression of fluorescence and determine the transfection efficiency.Mouse BMMSCs were purchased from Procell . The cells were maintained in DMEM/F-12 medium supplemented with 10% fetal bovine prostate tissue  at 37°C with 5% COThe cell viability of GFP-BMMSCs was measured using the Cell Counting Kit-8  according to the instructions of the manufacturer. Absorbance was recorded at 450 nm.Mice prostate tissues were fixed in 4% paraformaldehyde for 24 h and embedded in paraffin to a histopathological study by H&E stain. In brief, each mice prostate paraffin section was deparaffinized and dehydrated with xylene and graded ethanol. After staining with hematoxylin and eosin, the paraffin sections were washed with distilled water and dehydrated with graded ethanol and xylene. The inflammation degree of each prostate tissue was evaluated using a light microscope under 5 random fields.μg/sample) was separated via 10% SDS-PAGE. And then, the separated proteins were transferred to nitrocellulose membranes. The membranes were blocked with 5% nonfat dried milk overnight at 4°C and incubated with the following corresponding protein antibodies: TGFβ-RI , TGFβ-RII , phosphor (p)-Smad2/3 , Smad4 , Smad7 , and β-actin . Then, the membranes were washed with Tris-buffered saline/0.1% Tween (TBST) and incubated for 1.5 hours with HRP goat anti-rabbit IgG . The bands were visualized using the ECL system , and β-actin was used as an internal control. The net optical density was measured using Quantity One software (Bio-Rad).Prostate tissues were homogenized using a homogenizer with magnetic beads. Total protein was manufactured using RIPA buffer . The concentration of protein was determined by a BCA kit . Total protein (30 μm) were fixed in acetone for 10 min and rinsed 3 times with PBS. Then, the tissue sections were, respectively, incubated at 4°C overnight with rabbit anti-mouse CD29, CD44, and CD45 monoclonal antibodies . Sections were rinsed with PBS and incubated with bovine anti-rabbit IgG-FITC  for 30 min at room temperature. Finally, sections were incubated with DAPI and observed with a fluorescent microscope.IF stain was used to determine the expression of CD29, CD44, and CD45 in mouse prostate tissue. Mouse prostate paraffin sections  following the instructions of the manufacturer. The absorbance measured at 450 nm wavelength was estimated using an enzyme-linked immune monitor . The concentrations of TNF-α, IL-1β, CXCL2, CXCL9, CXCL10, CXCL11, MDA, SOD, and GSH-Px in the sample were calculated from the standard curve.The contents of TNF-t-test was used to assess statistical significance. Differences with a P < 0.05 were considered to indicate statistical significance.The data were represented as mean ± standard deviation. Statistical analysis was performed using SPSS 20.0 (IBM Corp.). One-way analysis of variance (ANOVA) with Tukey's post hoc test of means was used for multiple group comparisons. For comparisons between two groups, Student's unpaired As shown in As shown in Figures α and IL-1β in prostate tissue of CBP mice, which was inhibited by cotreatment of DHA and BMMSCs  and prostate cancer . Thus, wSeveral clinical studies demonstrated oxidative stress played an essential role in the progression of CP –24. To cβ-RI, TGFβ-RII, p-Smad2/3, and Smad4 was markedly increased  fluid and pulmonary inflammatory response by secreting antibacterial peptide hCAP-18/LL-37 [Earlier studies showed that mesenchymal stem cells (MSCs) played the role of inhibiting the inflammatory response through inhibiting T cell, B cell, and antigens that presented cell proliferation and suppressing proinflammatory factor generation . MSC conpression . Meanwhipression . In addicapacity . Moreovecapacity . In the 18/LL-37 . However18/LL-37 . Importaα, IL-1β, and chemokines CXCL2, CXCL9, CXCL10, and CXCL11. TNF-α is an important proinflammatory cytokine secreted by mononuclear macrophages, neutrophils, and T cells [β promoted the release of protein hydrolases from neutrophils and activated T cells to accumulate in the prostate tissue, finally causing an immune response [α and IL-1β levels were significantly higher in prostatic secretions of patients with CP [β, TNF-α, and IL-6 via regulating NF-κB signaling in acute lung injury (ALI) [We then investigated the effect of cotreatment with DHA and BMMSCs on inflammation in CBP mice. This study demonstrated that high-dose DHA and BMMSC cotreatment markedly diminished inflammation in prostate tissue of CBP mice, which was manifested as decreases in the expression levels of proinflammatory factor TNF- T cells . IL-1β presponse . The stu with CP . Moreove with CP . DHA wasry (ALI) . Moreovery (ALI) . DC32, ary (ALI) . These sStudies have shown that the development of CP was also closely related to oxidative stress , 40. CBPβ/Smad signaling played critical roles during inflammation and oxidative stress [β, TGFβ-RI, and TGFβ-RII phosphorylated Smad2/3 proteins and ultimately initiated transcription of the relevant target gene with the cooperation of Smad4 [βRI could hinder the activation of Smad2/3 and control TGF-β signaling in a negative feedback manner [β/Smad signaling pathway has been long considered as a key mediator in the suppression of inflammation, such as in renal inflammation [β/Smad signaling ameliorated oxidative stress and inflammation in a liver fibrosis model [β/Smad signaling pathway in prostate tissue of CBP mice. These opposite results may be due to the interaction of TGF-β/Smad with other cytokine signaling in the inhibition and promotion of inflammation [Previous reports have suggested that TGF-e stress , 44. Of of Smad4 . On the k manner . TGF-β/Sammation  and rheuammation . Howeverammation .β1/Smad signaling pathway. The combination therapy of DHA and BMMSCs has shown excellent promise in the treatment of CP.In conclusion, DHA performed potent anti-inflammatory and antioxidant effects in CBP mice by promoting the accumulation of BMMSCs into the prostate tissue. Meanwhile, the homing of BMMSCs promoted by DHA inhibited the activation TGF-


Csp), energy density (Ed), power density (Pd) and retention capacity (%) of the reverse micelles were estimated to be 584 F g−1, 143 W h kg−1, 2356 W kg and 97.5%. After magnetite incorporation, we achieved a maximum supercapacitive performance of 631 F g−1, 204 W h kg−1, 4371 W kg−1 and 98%. We demonstrate that the use of magnetite incorporated St–PBS reverse micelles minimizes the contact resistance between the two supercapacitor electrodes, resulting in high charge storage capacity.In this work, we report on a reverse micellization approach to prepare uncarbonized starch and poly hybrids with exceptional charge storage performance. Uncarbonized starch was activated through protonation, hybridized with poly , configured into conductive reverse micelles, and incorporated with magnetite nanoparticles. Before magnetite incorporation, the maximum specific capacitance  hybrids with exceptional charge storage performance. Conventional fossil fuels can be difficult to handle, are not renewable, disrupt local ecologies, are associated with various geo-political problems, pollute the environment and are increasingly being depleted at faster rates.3,4 Solar, wind, biofuels, hydrothermal are examples of renewable and sustainable energy systems that are presently gaining significant worldwide attention. However, the energy generated from these renewable resources must be stored in portable or stationary devices to ensure cleaner and smarter, and more accessible energy.5,6The process of employing energy systems to generate, convert and store energy needs to meet various sustainability indices, and the energy systems used should meet environmental, economic and social quality requirements.7–10 These carbon based electrodes have also been obtained from fish gills, corn syrups; but there has been limited research directed towards biopolymers like starch, poly(lactic acid), alginates, cellulose, chitosans and many more, for energy storage in supercapacitors. The few works reported on the use of starch, cellulose and other biopolymers shows that they are first converted to carbon forms before they are utilised in energy storage devices. This principle is a bit debatable, since carbonization itself makes use of high temperature and can utilize harsh solvents, which reduces the overall sustainability potential of the biopolymer materials in the long-run.Various electrodes that promise to meet various sustainability goals for energy storage include carbon nanomaterials from biomass such as coconut husks, bamboo, sugar-cane, banana peels, cocoa pods, rice husks, almonds, palm kernels and many more.11–13 Apart form the metal oxides, other hybrids of natural polysaccharides with several biodegradable polymers and conductive polymers have been reported.14–17 Conductive polymers like poly, polyaniline, and polypyrrole have shown similar or better pseudocapacitve capabilities than metal oxides for energy storage applications.18–22This work designed and implemented a unique synthesis and assembly protocol to develop an enhanced synergy between ‘uncarbonized’ biopolymers and nanomaterials to fabricate sustainable supercapacitor electrodes. The idea behind this work, is that the functional groups on the selected biopolymer can link easily with nano-oxides of transition metals to obtain suitable physico-chemical properties that are viable for energy storage. The use of uncarbonized starch portrays good sustainability and leverage on the biocompatibility, functionality and renewability of starch.23 Micelles or reverse micelles attains spherical, ellipsoidal, cylindrical and bilayer conformations, and different conformations shows different diffusive, conductive and ionic properties,24–26 which may affect their electrochemical properties in super-capacitor applications. In addition, different micellar designs or conformations show differing surface charge distribution that stabilizes their cores.24,27 The three different reverse micelles designed in this work showed different supercapacitve properties and magnetite nanoparticles were then incorporated within their cores to enhance free flow of ionic charges.The uncarbonized starch was activated through protonation to improve its electrochemical properties, and hybridized with poly (PBS), a semicrystalline biopolymer to make a hybrid biopolymer–polysacharide composite. The hybrids formed were configured into conductive reverse micelles, starch formed the head and PBS formed the tail.et al., reported on the design of nitrogen-doped carbon based non-nano micellar structures with specific capacitance of 271 F g−1. Sun et al., also reported on the synthesis of carbon aerogels that rearranges into spherical micelles with tunable porosity in the presence of [C16Im]BF4. The specific capacitance obtained was 188 F g−1, with a corresponding energy and power densities of 9.08 W h kg−1 and 6250 W kg−1. Till now, and to the best of our knowledge, no public research has so far been published on the use of the micellar forms of uncarbonized starch and PBS nano-hybrids for applications in supercapacitors.20There are very few works on the application of micelles for supercapacitor applications. However, there are numerous reports on the use of carbon aerogels, materials that can be synthesised using micelle based soft templates, which have not produced very high specific capacitances and energy densities. Liu 28,29 The switching of the conformations of these micellar nano-architectures can therefore be used to tune the conductance, capacitance, energy and power densities of supercapacitor electrodes for the storage of electrical charges.28–30 For example, this method is applied in conductive electrophoretic image displays, electrorheological fluids, and ink jet imprintings.29–32Different micelles orients in a way to increase their overall ionic stability. By carefully positioning metal oxides within micellar forms of uncarbonized biopolymer, excellent charge transfer kinetics can be achieved, and this can form the basis of how starch can be used as a supercapacitor electrode without converting it into a carbon form.Therefore, the aim of this particular work is to primarily use a green method to specifically design different sustainable micellar nano-architectures based on poly cores, and uncarbonized starch heads with impregnated nano-magnetite. The effect of each micellar design on the overall electrochemical conductivity and capacitive performance of starch–PBS nanohybrids will be studied. The novelty of our work is the use of different micellar designs to tune the supercapacitve properties of an uncarbonized starch based supercapacitor electrode.2.3·6H2O), ferrous chloride tetra-hydrate (FeCl2·4H2O), glass fibre separator, nickel foam, platinum wires, citric acid, p-toluene sulphonic acid, ammonium chloride, potassium chloride, acetonitrile, isopropanol, aqueous ammonia, oleic acid, Tween-80, dichloromethane (DCM), tergitol were obtained from the local supplier for Sigma Aldrich Pty Ltd.Materials used for this work; which include corn starch, poly (PBS) extended with 1,6-diisocyanato hexane, ammonium persulphate, ferric chloride hexa-hydrate  were made into three different types of reverse micelles and labelled as St–PBS micelle I, II and III. The first reverse micelle was designed using the precipitation method. In this method, 1.2 g of the prepared St–PBS hybrid was dissolved in 15 mL acetonitrile under room temperature using a magnetic stirrer at 250 rpm. The mixture was introduced into 25 mL of 5% tween-80 solution and further stirred for 6 hours at 90 °C, to ensure the complete evaporation of acetonitrile. The obtained gel was pre-set in a refrigerator at 4 °C for 24 hours, vacuum-dried under a pressure of 720 mmHg for 5 hours and weighed. The drying was done until two consecutive weights matched; indicating equilibrium was reached within the reverse micelle.35.The second reverse micellar design was designed using the single emulsion technique. In this technique, 1.2 g of the prepared St–PBS hybrid was dissolved in 20 mL, 1 : 1 dichloromethane and acetonitrile, under room temperature using a magnetic stirrer at 250 rpm. The mixture was introduced into 25 mL of 5% tween-80 solution and further stirred for 6 hours at 90 °C, to ensure the complete evaporation of acetonitrile and dichloromethane. The mixture was pre-set and dried under the same condition for the first reverse. The third micellar design was fabricated using the double emulsion method. Samples were typically prepared by dissolving 1.2 g of the prepared St–PBS hybrid was dissolved in 20 mL, 1 : 1 dichloromethane and acetonitrile, under room temperature using a magnetic stirrer at 250 rpm. The mixture was introduced into 15 mL of 5% tween-80 and 10 mL of 3% tergitol and stirred for 6 hours at 90 °C, to ensure the complete evaporation of acetonitrile and dichloromethane. The mixture was pre-set and dried under the same condition for the first reverse. All methods were modified and adopted from the ref. 2.4.p-toluene sulphonic acid on a magnetic stirrer at 70 °C for 30 minutes, to completely reach homogeneity. The acidity of the mixture was adjusted to reach a pH of six. Immediately, 0.05 g of magnetite nanoparticles was introduced into the mixture and stirred for 60 minutes in a three-necked flask, at 50 °C under reflux. Powder products were obtained through centrifugation, washed and pre-set under refrigeration at 4 °C. The final product was air dried.36A weighed amount (0.1 g) of the prepared St–PBS micelle I, II and III were differently dispersed in 5% solution of tetrahydrofuran and 2.5.−1. X-ray diffraction (XRD) patterns were obtained on a Rigaku Ultima IV X-ray diffractometer, employing CuKα radiation at a wavelength of 1.5406 Å (generated at 45 kV and 40 mA). XRD patterns were collected in the 2θ range between 5° and 90° with a step size of 0.01°, and a scan speed of 1° min−1. The particle sizes of the nano-hybrids were investigated using a Dynamic Light Scattering (DLS) instrument; Zetasizer, model ZEN 3600. The morphologies of the samples were determined using scanning electron microscopy (SEM),  at a 20 kV electron acceleration voltage. The surfaces of the samples were coated with carbon to avoid charging.Samples were analysed on a Fourier Transform Infrared (FTIR) Spectroscope equipped with an ATR accessory . The FTIR analysis was carried out in the wave number range of 4000 to 600 cm2.6.2SO4 electrolyte, respectively. The working electrode was prepared by loading on nickel foam; a 10 mg mixture of St–PBS micelles and poly(vinylidene difluoride) powder dissolved in N-methyl pyrrolidone in 80 : 20 by weights. The loadings were achieved through doctor blading, using a specially designed smooth and thin glass. Cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) measurements were performed with potentials ranging from 0 to 0.7 V. Electrochemical impedance spectroscopy (EIS), was conducted over a frequency range of 100 kHz to 0.1 Hz with perturbation amplitude of 0.01 V.37–40 In the two-cell configuration, which involves the working electrode and counter electrode set-up, a glassy fibre separator was soaked in 1 M Na2SO4 electrolyte, sandwiched in between two symmetrical working electrodes. The two-cell working electrodes were prepared using the same method employed for the three-cell working electrodes. The symmetrical supercapacitor was aligned, pressed and clipped before setting it up for two-cell electrochemical measurements.The conductivities of the St–PBS micelles were determined using a four-probe system, while their electrochemical characteristics were investigated on a Gamry potentiostat in both three and two cell configurations. In the three cell configuration, a platinum wire, an Ag/AgCl (saturated by 3 M NaCl), and St–PBS-magnetite electrodes on nickel foam were used as the counter, reference, and working electrode in 1 M Na3.41 while PBS improves the recyclability of the micelles and help the micelle to store charges over a wide temperature window.42The St–PBS reverse micelles with high charge storage capacity were designed from starch, and poly(butylene succinate) biopolymers. The starch provides porous channels for easy transfer of charges within the micelles,3.1.−1. The successful design of the reverse micelles was confirmed from the differences between the OH vibration regions of the St–PBS hybrids before and after micellization. Before micellization, the St–PBS hybrids showed a diminished O–H band, broad C–H bands at 3430 cm−1 and 2870 cm−1.43 The intensities of bands at 1169, 1085 and 920 cm−1 attributed to C–O and C–C stretch with C–O–H contributions. The C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O adsorption band at 1710 cm−1 for PBS narrowed after micellization, indicating strong bond formation between the starch and poly(butylene succinate) polymers.−1. Between 1300–900 cm−1, C–O–C glucosidic bonds, C–O–H bending and stretching vibrations, and C–O stretches were observed, and duplet C–H bends at 810 and 802 cm−1. St–PBS micelles I and II showed a more resolved band than St–PBS micelle III around 1050 to 950 cm−1; suggesting that St–PBS micelles I and II have shorter-ranged double helices than micelle III.44 Changes and shifts in the peak positions between 1300–900 cm−1 can be used to probe interactions between the starch and PBS.40,44,45 In general, the peaks shifted to slightly lower wavenumbers when comparing St–PBS micelles I  and II , which suggests an increase in hydrogen bonding between the starch and PBS components.46After micellization, the O–H stretching vibrations of St–PBS micelles I, II and III shifted to 3502, 3505 and 3511 cmθ angles of 19.7°, 22.2°, 26.34° and 29.10°, which are attributed to the (020), (110), (1̄21), and (111) reflections from the α crystal of the PBS component within the hybrids.47 When mixing starch and PBS, some authors have noted that the low intensity and relatively broad peaks of the starch molecules will not be observed in the composite, due to the mixing of the starch and with the much more crystalline PBS.23 However, we did observe some minor diffraction peaks in the three starch micelles composites, St–PBS micelle I–III, from 46° to 53°, which suggests the micellization process improves the crystallinity of the composite. From the most intense 2θ peak at 29.10°, the ‘d’ spacings from Scherrer equation for the St–PBS micelle I–III samples were 23.24, 19.38 and 26.17 nm respectively. The d-spacing values confirmed that the St–PBS micelle samples were relatively well-ordered with some crystallinity in the structures, and domains are in the nanometre range.The XRD analysis of the St–PBS hybrids , reveale3.2.48 The three reversed micelles had surface tensions of 50.23 ± 0.2, 58.14 ± 0.2 and 62.69 ± 0.1 N m−1; implying their surfaces are favourable for wetting and will coat well on the current collectors, and should allow for excellent ingress of the electrolytes.The three reversed micelles showed contact angles of 72.49°, 74.81° and 75.01°, which suggests good wetting properties.−1 was obtained at 373 K, for St–PBS micelle I, II and III respectively. This implies that the St–PBS micelles II sample should possess better capacitive properties, and maybe more stable when voltage is applied during electrochemical testing. Temperature increase creates distortion, which in turn, creates polarons and bipolarons movement within the chains of the starch micelles that had already been doped with para-toluene sulphonic acid. The flow of polarons and bipolarons is what is responsible for the conductivity of the designed micelles.The average size of the St–PBS reverse micelle samples varied from 70–90 nm, with no apparent trend in the variation of the sizes as determined by DLS . The con3.3.40,49 The differences in morphology are expected to have an effect on the supercapacitor properties due to differences in charge transfer and storage.The SEM images presented in 3.4.3.4.1.Csp) of the hybrids before and after micellization were calculated from the areas of CV curves  was used to investigate the capacitive behaviours of the three reverse micelles designed. The specific capacitances ,55 development of polypyrrole cellulose hydrogels (maximum Csp of 255 F g−1),56 and a PEDOT lignin poly(aminoanthraquinone) composite (maximum Csp of 418 F g−1).57At high scan rates, diffusion resistance and polarization phenomena increase, and hence the Rs), charge transfer resistance (Rct), and Warburg impedance (W) were obtained from Nyquist plots of the micelles  equivalent circuit , St–PBS micelle I (curve b), St–PBS micelle II (curve c) and to the St–PBS micelle III (curve d), highlights how the synthetic method improved the ideal capacitive behaviour of the materials. All of the three St–PBS reverse micelles completed their charge–discharge cycles faster than the ordinary St–PBS hybrid. The ordinary St–PBS hybrid completed its GCD cycle after 1400 seconds while St–PBS micelles I, II and III completed theirs in shorter periods of 800, 600 and 400 seconds respectively. This highlights that the micellization process not only affects the morphology, as seen with the SEM images, but also improves the charge and discharge kinetics of un-carbonized starch. The retention capacity of the St–PBS hybrid  of the St–PBS was 73 W h kg−1, at a scan rate of 5.0 mV s−1. After micellization, the St–PBS micelles I, II and III showed maximum values of 79, 143 and 77 W h kg−1 respectively at a scan rate of 5.0 mV s−1. The corresponding power densities for the ordinary St–PBS was 1640 W kg−1, and the St–PBS micelles I, II and III had power densities of 2118, 2356, 2254 W kg−1 respectively. Discussions on the significance of these results are presented in comparison to the samples tested in a two electrode cell configuration.The energy densities of the St–PBS hybrids are presented in 3.4.2.Csp and retention capacity were determined. The CV's were recorded from 0.0 to 0.4 V using 1 M Na2SO4 electrolyte, at scan rates of 5, 10, 20, 50, 100 and 200 mV s−1, and are presented in Csp of 101, 126, 144, 152, 170, and 182 F g−1. At the same scan rates, St–PBS micelle II showed Csp of 106, 140, 159, 181, 185, 203 F g−1, and St–PBS micelle III showed Csp of 103, 137, 155, 172, 183, 194 F g−1. These specific capacitances are lower than the ones reported for three-electrode configuration experiment  with the St–PBS micelle II sample.n’ values of 0.690, 0.74 and 0.70 were obtained; while an equivalent series resistance (ESR) of 1.97, 2.92, and 2.90 ohms were obtained for St–PBS micelles I, II and III respectively. Rct of 19.32, 18.17 and 19.08 ohms  were more intense, than the C–H peaks found in St–PBS micelle I with magnetite (St–PBS-I–Fe3O4) and the St–PBS micelle II with magnetite (St–PBS-II–Fe3O4). Carbonyl peak at 1635 cm−1 confirms the presence of starch biopolymers with reduced end groups, and C–O–C bond of the starch was observed at 1055 cm−1.65 The XRD patterns 3O4 nanoparticles.66 The peak at 2θ of 37° indicates the (311) crystal plane of magnetite nanoparticles, while the diffraction peaks at 2θ = 19.1° and 24.3° indicates the crystalline structure of starch biopolymers. The peak at 42.5° shows the (400) crystal planes of gamma magnetite nanoparticles, which are obtained at low temperature conditions.Magnetite nanoparticles have interesting physical–chemical properties that can be exploited to enhance the energy densities of the St–PBS micelles. They have multiple redox states, high thermal conductivity, and shows sharp conductance transitions.3O4 micelles appeared as agglomerated particles, with irregular spherical morphology. The morphology observed was similar to what has been reported in the literature.67–69The SEM images of the three magnetite incorporated reverse micelles showed similar morphologies Fig. S2. The St–3O4, while, the magnetite nanoparticles appeared as dispersions in the St–PBS-II–Fe3O4 and St–PBS-III–Fe3O4 samples. Selected Area Electron Diffraction (SAED) showed crystalline ring patterns, which were indexed with respect to inter-planar spacing of incorporated magnetite nanoparticles. Besides the (311) and (400) crystal phases, which were also observed with the XRD analysis, the SAED pattern for the St–PBS-I–Fe3O4 had rings indexed at (533), and (111), the St–PBS-II–Fe3O4 had an additional ring indexed to (220), and the St–PBS-III–Fe3O4 showed a ring indexed to (440) crystalline phase for magnetite. Similar results have been reported in the literature for magnetite starch composites.70 The Energy Dispersive X-ray (EDX) results confirmed that the three magnetite incorporated reverse micelles contained C, O and Fe with other minor peaks like Na, Cl and S peaks, from p-toluene sulphonic acid activation treatments.TEM images of the St–PBS micelles with the magnetite incorporated are presented in 3.6.−1, St–PBS-I–Fe3O4 had Csp of 86, 173, 288, 435, 527, and 608 F g−1, while St–PBS-II–Fe3O4 had Csp of 108, 172, 309, 442, 532, and 631 F g−1 respectively. The third magnetite incorporated micelle, St–PBS-III–Fe3O4 had Csp of 79, 154, 278, 393, 484, and 589 F g−1. Similar trends seen with the St–PBS micelle samples before magnetite incorporation were observed with samples with magnetite. At a scan rate of 5 mV s−1, the highest Csp values were observed, and the relative increases were (at 5 mV s−1) 87.7, 8.05, and 85.2% for the St–PBS micelle I, II, and III samples before and after magnetite incorporation. The increase in Csp values could be attributed to some changes in morphology, as noted with SEM images, and an increase in charge storage sites due to the interfaces between the magnetite nanoparticles and the St–PBS micelle hybrids.The samples with magnetite nanoparticles incorporated into the reverse micelles were then investigated using CV, EIS and GCD using both two and three electrode configurations. CV results using the three electrode configurations are presented in Csp observed with our work is greater than some values reported in the literature. For example, in the work reported by Rhadakrishan, where polypyrrole–magnetite and polyaniline–magnetite were designed, the largest Csp reported was 210 F g−1.71 Wang et al., obtained a Csp of 220 F g−1 after he incorporated magnetite nanoparticles in graphene, Oh et al., achieved an increase in Csp from 99.4 to 202 F g−1 after magnetite incorporation.31,72The increase in −1, the three magnetite incorporated St–PBS reverse micelles had energy densities of 148, 154 and 146 W h kg−1. The aim of magnetite incorporation is to improve the energy density of the starch based micelles. The relative increase in energy densities of the reverse micelles after magnetite nanoparticle incorporation was 87.3, 7.7, and 89.6% for the St–PBS micelle I, II, and III respectively. Energy density shows how much energy a supercapacitor electrode can store, and it provides an indication on where the supercapacitor can be applied.As shown in 3O4 had a power density of 2118 W kg−1, the St–PBS-II–Fe3O4 had a power density of 4371 W kg−1, and the St–PBS-III–Fe3O4 had a power density of 2356 W kg−1. Sevilla et al., reported a Csp of 200 F g−1 after activation of biomass with sodium sulphate; Sudhakar and Kumar,73 obtained a Csp of 115 F g−1 for starch doped with poly(styrene sulfonic acid), while Han et al.,74 reported a volumetric capacitance of 584 F cm−3 for starch activated with poly(4-styrene sulfonate). In our method, we employed para toluene sulfonic acid and propylene carbonate in the presence of chloride ions to activate our starch based micelles. Our work achieved specific capacitance and energy density values, which are greater than the obtainable values in current literatures, for starch based supercapacitors.Due to the high energy densities of these magnetite incorporated starch micelles, some potential areas of application include electro-rheological fluids, and in LED power sources. Finally, the St–PBS-I–Fe−1, the St–PBS-I–Fe3O4 had Csp of 107, 164, 183, 237, 277, and 273 F g−1; similarly, St–PBS-II–Fe3O4 showed Csp values of 124, 168, 218, 263, 295, and 308 F g−1 and the St–PBS-III–Fe3O4 had Csp of 109, 160, 202, 241, 284, and 291 F g−1. The energy density of the three respective micelles at 5 mV s−1 were 66, 79, 68 W h kg−1; and the power densities were 3118, 3291, 3157 W kg−1. The values for Csp and Ed for the two- and three-electrode configuration are summarised and compared in Tables S23O4 had a retention capacity of 96.0%, the St–PBS-II–Fe3O4 had 97.5% and St–PBS-III–Fe3O4 had 97.0%, after 5000 cycles  shows the materials have excellent potential for scale-up and device assembly.A new route, which is cost effective and environmentally friendly, was employed to design conductive un-carbonized starch for energy storage. Three different methods were employed to make three reverse micelles of St–PBS hybrids, the PBS providing strength and higher operational temperature window for the hybrid reverse micelles. The reverse micellar starch hybrid prepared using a single emulsion technique, St–PBS micelle II, produced agglomerated structures with some porosity that could account for the excellent capacitive properties observed. The St–PBS micelle II had the highest Csp of 631 (three-electrode test) F g−1 and 308 F g−1 (two electrode testing), and Ed of 154 (three electrode test) and 79 (two electrode test) W h kg−1. The significance with the values reported in this work is that all the samples were in un-carbonized natural polymers hybrids. Furthermore, this research showed that different micellar designs exhibit different ionic and electronic path properties leading to variations in conductivity and capacitance. Thus offering new routes in the design and implementation of natural un-carbonised polymers for energy storage. The reverse micelle II, which was designed using a single emulsion technique, showed the best supercapacitive properties in both two and three cells configurations. This is attributed to the unique stable architecture, and enhanced surface and charge properties. The constant phase element (CPE) which was used to model the surface kinetics of the St–PBS based micelles showed that they are good platforms for supercapacitive energy storage. Non-toxic, chemically stable, affordable and available materials were used throughout this work. The findings will provide alternatives in the science and engineering of cleaner and sustainable energy storage systems. Thus, with our approach, un-carbonized biopolymers can contribute to meeting the United Nations 7th sustainable development goal of ‘Affordable and Clean Energy’.Magnetite nanoparticles were incorporated into the micelles to achieve a cycling stability of 98% after 5000 cycles, a maximum There are no conflicts to declare.RA-011-D1RA00635E-s001