Staphylococcus aureus (3.2–log CFU mL−1 reduction) and were non-cytotoxic to human keratinocytes (HaCaT cells). The in vitro release profile of DEX was prolonged with the increasing number of layers, and the time-dependent data imply a diffusion/swelling-controlled drug release mechanism. In addition, the in vitro wound healing assay demonstrated a good cell migration capacity, headed to a complete gap closure after 24 h. These results certify the potential of these multi-layered polysaccharides-based patches toward their application in wound healing.Antibacterial multi-layered patches composed of an oxidized bacterial cellulose (OBC) membrane loaded with dexpanthenol (DEX) and coated with several chitosan (CH) and alginate (ALG) layers were fabricated by spin-assisted layer-by-layer (LbL) assembly. Four patches with a distinct number of layers  were prepared. These nanostructured multi-layered patches reveal a thermal stability up to 200 °C, high mechanical performance (Young’s modulus ≥ 4 GPa), and good moisture-uptake capacity (240–250%). Moreover, they inhibited the growth of the skin pathogen  Natural polymers are being explored as building blocks to engineer multifunctional sustainable materials with sophisticated architectures via biomacromolecular assembly strategies. Among the prevailing methodologies to design such materials, the layer-by-layer (LbL) assembly is a simple, versatile and well-established technique with high relevance in the drug delivery domain . In factPolysaccharides are amid the most commonly utilized components for the design of multi-layered systems applied on drug delivery, despite the constraint of not yielding robust and free-standing films or capsules without the use of a template, either sacrificial or non-sacrificial  . In the Our interest in developing multi-layered BC derived patches by LbL assembly for cutaneous drug delivery originates from the idea of combining the peculiar properties of a freestanding exopolysaccharide robust membrane  with the properties of hydrophilic ionic polysaccharides  and alginate (ALG)), and an API with wound healing potential  used in several pharmaceutical formulations in the field of dermatology and skin care). Although BC (either in its pure or oxidized forms) has already been individually blended with CH/ALG ,10 or DEStaphylococcus aureus, the in vitro cytotoxicity toward human HaCaT keratinocyte cells and the wound healing potential.In this perspective, the goal of the present study is to design multi-layered patches composed of three polysaccharides, namely BC, CH, and ALG, as antibacterial cutaneous drug delivery vehicles for DEX, viz. a water-soluble API with wound healing ability. The multi-layered patches with a stratified structure were assembled via spin-assisted LbL methodology using a DEX-loaded OBC membrane as the initial negatively charged substrate, followed by the alternate adsorption of positively charged CH and negatively charged ALG polyelectrolytes, in a total of 5, 11, 17, and 21 layers. An in-depth analysis of the structure, morphology, thermal stability, mechanical properties, and moisture-uptake capacity of the multi-layered patches is presented, as well as the in vitro drug release profile at physiological pH, the in vitro antibacterial activity against ® . Type 1 ultrapure water (resistivity of 18.2 MΩ cm (25 °C)) was filtered by a Simplicity® Water Purification System . Other chemicals and solvents were of laboratory grade.Dexpanthenol , glycerol (≥99.5%), phosphate buffered saline , sodium hypochlorite solution (available chlorine 10–15%), sodium bromide (≥99%), sodium alginate ), 2,2,6,6-tetramethylpiperidine-1-oxyl , tryptic soy broth (TSB), tryptic soy agar (TSA), 3--2,5-diphenyltetrazolium bromide , and dimethyl sulfoxide  were supplied by Sigma-Aldrich . Dulbecco′s Modified Eagle′s Medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin/streptomycin, and fungizone were purchased from Gibcov/v) aqueous acetic acid solution, filtered, and precipitated by neutralization with NaOH 0.5 M up to a pH of 8.5. The precipitate was rinsed with water until neutral pH, followed by freeze-drying [Chitosan ) was obtained from Mahtani Chitosan Pvt. Ltd. India. For purification, CH was dissolved in a 1%  bacterium was received from American Type Culture Collection (ATCC) collection . The nontumorigenic immortalized human keratinocyte HaCaT cell line was acquired from Cell Lines Services .Bacterial cellulose (BC) was biosynthesized in our laboratory by the embranes . Staphyl−1) were stored in distilled water at 4 °C until further use.The oxidized bacterial cellulose (OBC) membranes were prepared via TEMPO-mediated oxidation as described elsewhere, with some modifications . Shortly−1) were prepared in ultrapure water and 1% (v/v) acid acetic aqueous solution, respectively. Wet OBC membranes (6 × 4 cm2) were weighted, drained, and then soaked in 5 mL of an aqueous buffered solution (pH 7.4) of DEX (0.15% w/v) with glycerol (1% w/v). The membranes were stirred at 100 rpm and 30 °C for 1 h to allow the complete absorption of the solution (viz. 100% entrapment efficiency) and then left to dry at 30 °C for 24 h. The DEX-loaded OBC patch was coated with 250 μL of CH (zeta (ζ)-potential: 57 ± 3 mV ) and 250 μL of ALG  aqueous solutions by spin coating  at 2000 rpm during 15 s. A total of 5, 11, 17, or 21 layers of the polysaccharides were deposited, starting and finishing with CH, as compiled in First, ALG and CH solutions . All measurements were executed at fifteen different locations of the specimens, randomly selected, with an accuracy of 1 μm.®, London, UK), in the wavenumber range of 600–4000 cm−1 at a resolution of 4 cm−1 over 32 scans.Attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra were computed on a Perkin-Elmer FT-IR System Spectrum BX spectrophotometer  coupled with a single horizontal Golden Gate ATR cell (SpecacScanning electron micrographs (SEM) were recorded in a HR-FESEM SU-70 Hitachi microscope . The specimens were placed on an aluminum plate and formerly coated with a carbon film.−1 under a nitrogen atmosphere.Thermogravimetric analysis (TGA) was undertaken in a SETSYS SETARAM TGA analyzer . The specimens were heated from 25 °C to 800 °C at a heating rate of 10 °C min−1 using a 500 N static load cell and gauge length of 30 mm. The specimens were rectangular strips (5 × 1 cm2) dried at 40 °C. All measurements were held on seven replicates.Tensile mechanical tests were conducted on a uniaxial Instron 5966 testing apparatus  in traction mode at a deformation rate of 10 mm min2) in a conditioned cabinet with 100% relative humidity at 25 °C for 22 h. After that period, the weight (wW) of the moist specimens was measured, and the moisture-uptake was determined according to the equation:W0 is the initial weight of the dry specimens [The moisture-uptake capacity was assessed by laying the dry specimens (2 × 2 cmpecimens .2) were immersed in a vessel containing 30 mL of 0.01 M PBS (pH 7.4) and the dissolution tests were then carried out at 32 °C and 50 rpm during 90 h. At regular time intervals, aliquots of 3 mL of each solution were withdrawn, and the same volume of fresh PBS was added to maintain a constant volume. The DEX concentration in each aliquot was determined by UV–Vis spectroscopy  at 206 nm with the linear calibration curve: 2 = 0.9978), in the range of 10–100 µm mL−1. The DEX content at each time was plotted as a cumulative percentage release concentration calculated using the formula:nC and nC−1 are the concentrations of DEX at time n and n − 1, respectively. Nine replicates were performed for each specimen [DEX-loaded patches . Each film specimen (4 × 6 cm2) was placed in contact with 5 mL of bacterial suspension via a ten-fold dilution of the overnight grown culture in PBS (pH 7.4). A bacterial cell suspension was tested as the control while OBC membranes, neat and DEX-loaded, were tested as blank references. All specimens were incubated at 37 °C under horizontal shaking at 120 rpm. At 24 h contact time, aliquots (500 μL) of each specimen and controls were collected and the bacterial concentration (CFU mL−1) was determined by plating serial dilutions on TSA medium. The plates were incubated at 37 °C for 48 h. The CFU were determined on the most appropriate dilution on the agar plates. Two independent experiments were carried out and, for each, two replicates were plated. The bacteria log reduction of the specimens was calculated as follows:The antibacterial activity of the OBC-based membranes was examined against −1 penicillin/streptomycin, and 250 µg mL−1 fungizone at 37 °C in 5% CO2 humidified atmosphere. Cells were observed daily under an inverted phase-contrast Eclipse TS100 microscope . The tests were performed in triplicates of OBC, OBC/DEX, and OBC/DEX/CH/ALG_21. Specimens of 1 × 1 cm² were prepared, sterilized by ultraviolet radiation, and then incubated with 1.0 mL of complete DMEM medium at 37 °C with 5% CO2 for 24 h to prepare the specimen extract.The cytotoxicity of the patches was evaluated in human keratinocytes cell line (HaCaT cells) by the MTT assay . BrieflyMeanwhile, one 96-well plate was prepared with 5 × 4 wells filled up with 6000 cells/well and the cells were then incubated in complete culture medium for 24 h for adhesion. After that time, cell culture medium (in the 96-well plates) was substituted by 100 μL of each of the extracts obtained from the incubated specimens and cells were then additionally incubated for either 24 h or 48 h. As a negative control, HaCaT cells were incubated with complete DMEM medium and treated identically as described for the specimens.−1) was added to each well and incubated for 4 h at 37 °C in a 5% CO2 humidified atmosphere. After that, the culture medium with MTT was removed and replaced by 150 μL of DMSO and the plate was positioned in a shaker for 2 h in the dark to totally dissolve the formazan crystals. The absorbance of the specimens was measured with a BioTek Synergy HT plate reader  at 570 nm with blank corrections. The cell viability was calculated with respect to the control cells: DMSO solvent; and At the end of the incubation time, 50 μL of MTT  of the scratched area of each condition were taken and compared to the corresponding 0 h time point.The effect of the multi-layered patches on the migration capability of HaCaT keratinocyte cells was evaluated using the scratch assay. Cells were seeded in a 6-well plate at 5 × 10p < 0.05.The statistical analysis was conducted with OriginPro 9.0.0 , where the statistical significance was assessed by the analysis of variance (ANOVA) and Tukey’s test with the statistical significance established at Polysaccharides-based multi-layered patches composed of OBC, CH, and ALG were fabricated by spin-assisted LbL coating assembly technology, as illustrated in 2 of OBC, selected based on equivalent commercial formulations [w/v, 2.1 mg per cm2 of membrane) was incorporated in the membrane as a plasticizer to upsurge their malleability and conformability to the skin surface [The OBC membrane was then loaded with DEX  of the positively charged CH  and the negatively charged ALG  polyelectrolytes were prepared c. CH andAll multi-layered patches are whitish and homogeneous, as displayed in the photographs of −1 (C–O stretching), 1316 cm−1 (O–H in plane bending), 2897 cm−1 (C–H stretching), and 3340 cm−1 (O–H stretching) [−1 assigned to the antisymmetric vibration of ionized carboxyl groups (νas(COO–), The modification of BC via TEMPO-mediated oxidation to obtain OBC with negatively charged groups was confirmed by ATR-FTIR spectroscopy through the presence of the typical absorption bands of the BC backbone at 1029 cmetching) , and the−1 (O–H and N–H stretching), 1636 cm−1 , and 1535 cm−1 (amide II band) [2 of OBC). In the SEM micrograph of the surface of the OBC/DEX patch  ,27 are nEX patch c, the na−1 (O–H stretching), 1638 cm−1 (C=O stretching and N−H bending), 1592 cm−1 (−NH2 bending), 1380 cm−1 (−CH2 bending), 1148 cm−1 (C−O−C antisymmetric stretching), 1078 and 1032 cm−1 (C−O stretching) [−1 (O–H stretching), and 1596 cm−1 (COO− antisymmetric stretching) and 1406 cm−1 (COO− symmetric stretching) [−1 and 1030 cm−1  and 3250 cm−1 (O–H stretching), [The ATR-FTIR spectra of the multi-layered patches b portrayetching) , and ALGetching) . MoreoveRegarding the morphology c, the SEThe thermal stability of the multi-layered patches was measured by thermogravimetric analysis under a nitrogen atmosphere. According to w/v)) into the OBC porous structure promoted a decrease in both Young’s modulus and tensile strength to 3.5 ± 1.2 GPa and 97 ± 15 MPa, respectively, complemented by an increase in the elongation at break to 4.9 ± 0.6%. This behavior is comparable to the trend obtained for pure BC membranes after the incorporation of APIs such as diclofenac [co-glycolic acid), poly(ethylene oxide), and poly(ɛ-caprolactone), loaded with DEX [The mechanical properties of the multi-layered polysaccharides-based patches were also analyzed, and the data obtained from the stress-strain curves are condensed in clofenac  and lidoclofenac  into thewith DEX .After the LbL assembly of the oppositely charged polyelectrolytes  on the negatively charged OBC substrate, the mechanical performance of the multi-layered membranes increased with the increasing number of layers. The Young’s modulus increased from 4.2 ± 0.8 GPa for OBC/DEX/CH/ALG_5 to 10.9 ± 4.0 GPa for OBC/DEX/CH/ALG_21, whereas the tensile strength increased from 142 ± 39 MPa for OBC/DEX/CH/ALG_5 to 188 ± 57 MPa for OBC/DEX/CH/ALG_21. On the contrary, the elongation at break decreased from 4.3 ± 0.7% for OBC/DEX/CH/ALG_5 to 2.2 ± 0.4% for OBC/DEX/CH/ALG_21, meaning that the membranes became less elastic as the number of layers increased from 5 to 21. Nonetheless, these hydrophilic multi-layered patches are satisfactorily malleable to conform to the surface of the skin.Worth noting is the fact that these OBC/DEX/CH/ALG multi-layered patches exhibited a higher tensile strength than the similar non-multi-layered films based on hydrogen peroxide oxidized BC (HOBC) or periodic acid oxidized BC (POBC) containing CH and ALG, which were prepared by blending the homogenized HOBC or POBC pulp with a CH and ALG gel solution, and studied for application as wound dressings .The moisture-uptake capacity of the multi-layered patches was determined to anticipate their interaction with moisture and exudates when applied to wound sites. Hence, the patches were left in a chamber with 100% relative humidity at 25 °C for 22 h. According to Therefore, any of these multi-layered polysaccharide-based patches can promote (if necessary) a moist environment at the skin interface and remove the surplus of exudates or other body fluids when applied to the skin surface.The in vitro release profile of the multi-layered polysaccharides-based patches was tested in PBS (pH 7.4) at 32 °C that simulates the usual pH and temperature of the skin . Figure In general, the release profile of the patches is quite standard, with an initial burst followed by a plateau where the DEX release achieves the highest value. While the OBC/DEX and OBC/DEX/CH/ALG_5 patches reached a maximum cumulative release of ca. 95% after 16 h, the OBC/DEX/CH/ALG_11 achieved ca. 87% after 16 h and OBC/DEX/CH/ALG_17 attained ca. 85% after 28 h. In the case of the OBC/DEX/CH/ALG_21 patch, it only reached a maximum cumulative release of ca. 65% after 90 h. This is a direct indication that the multi-layered systems retarded the DEX release from the OBC membrane, particularly for 21 layers. Moreover, this slow-release profile is suited for wounds that require longer therapeutic treatments combined with a local skin moisturizer with antibacterial properties and wound healing ability.When compared with non-multi-layered BC-based membranes loaded with water-soluble APIs ,8, thesen) of 0.45 was obtained for OBC/DEX, 0.68 for OBC/DEX/CH/ALG_5, 0.69 for OBC/DEX/CH/ALG_11, 0.68 for OBC/DEX/CH/ALG_17, and 0.70 for OBC/DEX/CH/ALG_21 (n < 1.0) , for the case of the multi-layered polysaccharide-based membranes [The in vitro drug release curves b were fuechanism ,39. BaseH/ALG_21 . These cembranes ,38,39.S. aureus (a Gram-positive bacterium) for 24 h, as shown in S. aureus, as previously reported [S. aureus was reduced by 0.8–log CFU mL–1 after 24 h. Although DEX is not an antibacterial agent [The antibacterial activity of the multi-layered polysaccharide-based patches was assayed against reported . Neverthal agent , it seemCFU mL–1 reduction after 24 h  as well as that of OBC and OBC/DEX was assessed in human HaCaT keratinocyte cells via the indirect MTT assay. This cell line was elected because it has been intensely used as a model for epidermal cells ,43,44,45With regard to the multi-layered patch with the higher number of layers , it was also deemed as a non-cytotoxic material because the cell viability was 95 ± 5% after 24 h and 97 ± 6% after 48 h b. FurtheS. aureus and non-cytotoxicity toward HaCaT cells, the next step focused on the wound healing potential of these patches as delivery vehicles for DEX. Therefore, the wound healing activity of the multi-layered polysaccharide-based patches with 11 and 21 layers (OBC/DEX/CH/ALG_11 and OBC/DEX/CH/ALG_21) as well as of the OBC/DEX patch was assessed using the in vitro wound healing scratch assay [Having proved that the multi-layered polysaccharide-based patches are robust, thermally stable, and present a controlled drug release profile, antibacterial activity against ch assay . TherefoOverall, and in the case of the patches, the HaCaT keratinocyte cells situated near the boundary of the scratched region moved toward the vacant region as time progressed, achieving a full closure after 24 h. Furthermore, the cell density at the back of the scratch site is also growing with time, which points to the proliferation of the HaCaT keratinocyte cells. However, in terms of temporal progression, the patches containing DEX, namely OBC/DEX, OBC/DEX/CH/ALG_11 and OBC/DEX/CH/ALG_21, demonstrated higher coverage of the wound-like gap area after 9 h, induced by cell growth and migration, when compared to the positive control. Despite the analogous behavior of the HaCaT keratinocyte cells after 24 h of exposure to the three patches , the optGiven that wound closure is a signal of wound healing ability , these aStaphylococcus aureus with 3.2–log CFU mL–1 reduction and are non-cytotoxic to human keratinocytes (HaCaT cells) with a cell viability of about 97% after 48 h. The in vitro DEX release profile from the patches is time-dependent and is prolonged with the increasing number of layers, and the in vitro wound healing assay showed a good migration capacity with the full occlusion of the wound scratch after 24 h. These data validate the potentiality of these antibacterial multi-layered polysaccharides-based patches as vehicles for the topical delivery of DEX, which is a promoter of wound healing.Functional multi-layered patches composed of OBC, CH, ALG, and DEX were prepared for wound healing applications. Four multi-layered patches with different number of layers, viz. 5, 11, 17, and 21, were assembled by the spin assisted LbL coating technique of oppositely charged CH and ALG polyelectrolytes onto the OBC cellulosic substrate loaded with DEX. These patches with a stratified structure had thermal stability up to almost 200 °C, mechanical properties with a Young’s modulus higher than 4 GPa, and good moisture-uptake capacity (240–250%). Additionally, they inhibited the growth of


However, manipulating the magnetic properties of nano-sized hematite , magnetic remanence (Mr), and coercivity (Hc)) can make them useful for nanomedicine  and nanoelectronics . Herein we study the effects of size, shape, and crystallinity on hematite nanoparticles to experimentally determine the most crucial variable leading to enhancing the radio frequency (RF) heating properties. We present the synthesis, characterization, and magnetic behavior to determine the structure–property relationship between hematite nano-magnetism and RF heating. Increasing particle shape anisotropy had the largest effect on the specific adsorption rate (SAR) producing SAR values more than 6 × greater than the nanospheres , indicating α-Fe2O3 nanorods can be useful for magnetic hyperthermia.Nano-sized hematite (α-Fe Althougreatment , 9, bulkreatment , 11 (i.e.3 emu/g ).An alternating magnetic field (AMF) causes magnetic particles to generate heat by way of four different mechanisms: eddy current heating, hysteretic heating, Brownian relaxation, and Néel relaxation . Bulk si3O4, γ-Fe2O3, and MFe2O4 where M = Ni, Mn, or Co [Control of nanoparticle size, shape, and crystallinity of superparamagnetic materials  can be n, or Co . Howevern, or Co , 15. Hemn, or Co , 9 instrn, or Co ) given an, or Co –22 existIn this work, we synthesized five different hematite samples with different sizes, shapes, and crystallinity to examine the structure–property relationship between hematite nano-magnetism and RF heating. Their wet-chemical syntheses were carried out following procedures previously developed, but their magnetic properties were not characterized , 24. We 2O3 rugby balls, nanospheres, and nanodiamonds, were ACS grade reagents purchased from Sigma Aldrich. For the α-Fe2O3 nanosheets and nanorods, all chemicals were purchased from Sigma Aldrich with purity All materials used for synthesizing the α-Fe2O3 particles were synthesized according to procedures outlined in previous works [4)3 was rapidly heated to 98 Rugby ball shaped α-Feus works . Succinc2O3 nanoparticles were synthesized according to procedures outlined in previous works [32O was added to 2 L of 98 Sphere shaped α-Feus works . In summ2O3 nanoparticles were synthesized according to procedures outlined in previous works [3 was heated to 98 3)3 · 9H2O was added under rapid mixing. The solution was then sealed to incubate for seven days.Diamond shaped α-Feus works . In shor2O3 nanoparticles were synthesized according to procedures outlined in previous works [4 · 7H2O was dissolved into the heated solution. After 2 h, the solution color changed from black to dark orange. The resulting precipitate was filtered out of the solution and washed with excess DI water. To remove the CuO hard templates, the precipitate was washed three times with 300 mL of concentrated ammonium hydroxide. The remaining orange sample was washed again with DI water, vacuum dried, and crushed into a powder. The crushed powder was then heated in air at 400 Sheets of α-Feus works . In summ2O3 nanorods were synthesized according to procedures outlined in previous works [42O was dissolved into 50 mL of DI water. Nitrogen gas (N2) was bubbled through each solution for 30 min to remove dissolved oxygen. Both solutions, the iron sulfate solution and the sodium hydroxide solution, were added together and allowed to react for 1 h under magnetic stirring with N2 bubbling at 50 2 gas, vacuum dried, and crushed into a powder. The crushed powder was then heated in air at 400 α-Feus works . 60 g ofMagnetization measurements of all materials were collected with a Superconducting Interference Device (SQUID) complete with a MPMS XL (Quantum Design Inc.). Magnetization curves from -10 kOe to 10 kOe at 300 K were collected for each nanoparticle morphology. For the zero-field cooled (ZFC) and field cooled (FC) curves, magnetic measurements were taken from 5 K to 300 K with a set magnetic field of 100 Oe. For each magnetic measurement the hematite samples were weighed and wrapped in Teflon tape.Each nanomaterial was analyzed by powder x-ray diffraction (XRD) using a Rigaku SmartLab X-ray diffractometer with Cu K TM) with an acceleration voltage of 2.5 or 5 kV.Dilute dispersions of the hematite samples in ethanol were drop-coated onto silicon wafers and imaged with the Helios 660 SEM/FIB microscope (Thermo Scientific. A 1 kW EASYHEAT induction heating system (Ambrell), was used to generate an alternating magnetic field at the fixed frequency of 325 kHz. The heating system consisted of a 5-turn coil with peak field strength 13.3 kA/m .Radio Frequency (RF) Heating experiments were performed using a slightly modified procedure previously used by others . A 1 kW To measure SAR values, each nanomaterial was dispersed in 1 mL of DI water  and placed inside of a 2 mL cryovial. The cryovial was then insulated with Styrofoam and placed into the center of the inductive coil. The induction heating system was then turned on for 90 s. The temperature of the solution was measured with a real-time fiber optic temperature sensor probe over time (LumaSense Technologies m3300), from which a linear fit of the average slope (dT/dt) of the first 20 s was obtained. The average slope of the solvent alone was subtracted to compensate for heat exchange with the surroundings. SAR was calculated with the following equation below:2O3 crystal structure through x-ray diffractometry.28 2θ of 24.2°, 33.2°, 35.6°, 40.9°, and 49.4° corresponding to α-Fe2O3, are shown in the XRD patterns in Fig. We confirmed the as-synthesized hematite nanoparticles had the α-Fes) values of 0.69 emu/g, 3.25 emu/g, and 4.58 emu/g, respectively  and magnetic remanence (Mr) values [r/Ms ratio = 0. The Mr/Ms ratio (which signifies the extent of ferromagnetism) of the nanospheres and the nanorods were 0.029 and 0.058, respectively, indicating they were ferromagnetic [r/Ms ratio (= 0.006), indicating that they were closest to acting superparamagnetic. In going from the larger rugby balls (~178 nm) to the smaller nanospheres (~66 nm), the magnetic hysteresis disappears, with magnetic remanence Mr (magnetization at zero magnetic field strength) and coercivity Hc (magnetic field strength at zero magnetization) values decreasing.The other hematite nanoparticle shapes  show superparamagnetic-like behavior, based on their very low coercivity (H) values . None armagnetic . The hemi.e., the curves do not overlap) due to particle-particle interactions [. The ZFC and FC curves of the nanodiamonds and the nanorods  and field cooling (FC) curves were collected at 100 Oe from 6 K to 300 K for all the nano-sized hematite samples Fig. . ZFC andractions . The ZFCods Fig. c, a alsom)—the temperature where the magnetic properties of hematite transition from weakly ferromagnetic to antiferromagnetic—occurs at 198 K and 209 K for the nanospheres and nanodiamonds  of ~57 K Fig. c. This m. Particles that did not present grain boundaries  were concluded to be single crystalline particles. For sufficiently small enough particles, only a single crystallite exists within the particle volume, which results in a single magnetic domain, the absence of hysteresis, and the inability to undergo hysteretic heating [The crystallinity of the samples was determined previously with SEM , 24 and  heating .The nanospheres, nanorods, and nanosheets were polycrystalline , 24, whiShape has a significant effect because elongating a nanoparticle increases its coercivity, magnetic remanence, and magnetic saturation values , 35–37, In a typical AMF heating experiment, a water suspension was placed in a 2-mL cryovial and placed within Styrofoam insulation. This was then placed inside a 1 kW EASYHEAT induction heating system (Ambrell) and an AMF was applied for 90 seconds at a fixed frequency of 325 kHz  . Suspensmeas was calculated using Equation 1 and from the slope of the experimentally determined temperature profiles of Fig. 3O4 nanoparticles  whic–200 W/g . The nanmeas value . More than twice as big, the rugby balls had a SARmeas value that was within error of that of the nanospheres despite having the largest magnetic hysteresis loop , the nanosheets were ~200 nm wide, which is too large to undergo Brownian/Neel heating and too small for eddy current heating. Thus, they undergo hysteretic heating, albeit with a low SARmeas value .The superparamagnetic-like nanosheets had the second lowest SARmeas value of all the hematite samples . By exploring the relationship between hematite structure  and its magnetic heating properties, we demonstrated that hematite nanorods can undergo radio frequency heating, generating appreciable SAR values that are 6 × greater than bulk hematite . They produce SARmeas values close to those suggested for magnetic hyperthermia treatment of tumours . Such fAdditional file 1. Additional figures and tables.


Persistent organic pollutants (POPs) are important environmental chemicals and continued study of their mechanism of action remains a high priority. POPs, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,7,8-tetrachlorodibenzofuran (TCDF), and polychlorinated biphenyls (PCBs), are widespread environmental contaminants that are agonists for the aryl hydrocarbon receptor (AHR). Activation of the AHR modulates the gut microbiome community structure and function, host immunity, and the host metabolome. In the current study, male C57BL6/J mice were exposed, via the diet, to 5 µg/kg body weight (BW) TCDF or 24 µg/kg BW of TCDF every day for 5 days. The functional and structural changes imparted by TCDF exposure to the gut microbiome and host metabolome were explored via 16S rRNA gene amplicon sequencing, metabolomics, and bacterial metatranscriptomics. Significant changes included increases in lipopolysaccharide (LPS) biosynthesis gene expression after exposure to 24 µg/kg BW of TCDF. Increases in LPS biosynthesis were confirmed with metabolomics and LPS assays using serum obtained from TCDF-treated mice. Significant increases in gene expression within aspartate and glutamate metabolism were noted after exposure to 24 µg/kg BW of TCDF. Together, these results suggest that after exposure to 24 µg/kg BW of TCDF, the gut microbiome increases the production of LPS and glutamate to promote localized gut inflammation, potentially using glutamate as a stress response. The relationship between the intestinal microbiome and the host metabolome is important to understand drug and xenobiotic metabolism, and mode of action ,2. Intesp-dioxin (TCDD), along with 2,3,7,8-tetrachlorodibenzofuran (TCDF) and coplanar polychlorinated biphenyls (PCBs), are agonists of the aryl hydrocarbon receptor (AHR) and have been reported to modulate the intestinal microbiome [CYP1A1, which is important for the metabolism of numerous xenobiotics [2,3,7,8-tetrachlorodibenzo-crobiome ,10,11,12crobiome ,14. AHR crobiome . The AHRobiotics . Since Aobiotics ,16.AHR-dependent changes in the microbiome can cause increased gut inflammation, which can lead to further intestinal dysfunction, thus worsening inflammation ,18. A maTCDF is an AHR agonist with reduced potency and half-life in vivo relative to TCDD but has a similar mode of action ,21. In tClostridium clusters XIVb and IV, which were both significantly decreased in the 24 µg/kg BW TCDF mice. Additionally, Oscilibacter and unclassified Ruminococcaceae in the 5 µg/kg BW TCDF, but these changes were not significant following 24 µg/kg BW TCDF exposure. Phyla shifts were not significant; however, trends seen in Bacterial population shifts seen in PICRUSt (Phylogenic Investigation of Communities by Reconstruction of Unobserved States) predicts the functional changes of the microbiome by examining taxonomical shifts in the microbiome and assigning known functions to the intestinal bacteria that increased or decreased in abundance. PICRUSt predictions are based on the functional capability of the microbiome rather than a direct examination of actively transcribed genes. After PICRUSt, 14 different pathways were predicted to be significantly upregulated or significantly downregulated after the 24 µg/kg BW TCDF treatment . Of partp = 0.04), glycine , proline , serine , leucine , threonine , and tryptophan  significantly increased after the 24 µg/kg BW exposure to TCDF. Additionally, there were significant decreases in UDP-N-acetyl-glucosamine  and N-Acetyl-glucosamine-1/6-phosphate  in the 5 and 24 µg/kg BW exposure to TCDF, respectively.All metabolites identified were then used for pathway enrichment through MetaboAnalyst ,27,28. TThe pathways represented by KEGG categories from the SAMSA2  analysis are illustrated in p = 0.0355, Q = 0.195).Pathway enrichment was performed using the metatranscriptomics data. The KEGG pathway for LPS biosynthesis shown in KEGG pathways for alanine, aspartate, and glutamate metabolism are illustrated in Glutamate catabolism has been shown to be an important indicator of bacterial stress . Within The non-significant change seen with glutamate, coupled with the significant increase of glutamate synthase gene expression, could be due to increased downstream glutamate utilization. This was supported in p = 0.031). An increase in LPS levels after TCDF exposure has been previously described [p = 0.038) after 24 µg/kg BW TCDF as compared to the control. This discrepancy is likely due to the significant decrease  in the total bacterial population seen in the cecal contents after treatment with 24 µg/kg BW TCDF , zonula occludens 1 (Zo-1), and zonula occludens 2 (Zo-2) in colon tissue. Both Zo-1  and Zo-2  were significantly decreased after exposure to 24 µg/kg BW TCDF. Previous research supports the above claim by noting that an altered intestinal microbiome composition can reduce the expression of Zo-1 and Ocln, both of which are responsible for creating tight junctions in the intestinal tissue [Zo-1 and Zo-2, results in the significant increase of gut permeability and circulating LPS in the serum.l tissue . Decreasl tissue . The depA Venn diagram  of signiOther than valine, leucine, and isoleucine biosynthesis and pathways with an unknown function, the PICRUSt results do not overlap with metatranscriptomics . MetatraElucidating the function of the intestinal microbiome remains a major milestone for drug discovery, risk assessment, and environmental toxicant research. We investigated the effects of 5 or 24 µg/kg BW exposures to TCDF on the mouse gut microbiome. Despite the limited taxonomic changes in the intestinal microbiome, there were large shifts in intestinal microbiome functionality after 24 µg/kg BW exposures to TCDF. Through metatranscriptomics, increases of LPS biosynthesis and the alanine/aspartate/glutamate metabolism pathways were identified. Data from metabolomics and LAL assays were integrated with the metatranscriptomic results and supported an increase of LPS biosynthesis. LPS was significantly decreased in cecal contents, likely due to a depleted gut bacterial population and an increase of gut permeability as LPS was significantly increased in the serum after 24 µg/kg BW exposures to TCDF due to increased LPS biosynthesis, coupled with the increase of gut permeability. These results support a potential relationship where strong AHR agonists like TCDF can induce LPS biosynthesis in the intestinal microbiome while simultaneously increasing gut permeability and result in an increase of circulating LPS in the serum. Overall, additional investigation into the in vivo relationship between bacterial-derived LPS, sustained activation of AHR by TCDD, and host immunity is warranted.n = 18) were acclimatized for one week and then underwent one week of pill training. Mice were then fed untreated dough pills (n = 6) or dough pills containing 5 µg/kg BW (n = 6) or 24 µg/kg BW (n = 6) TCDF for five consecutive days (one pill per mouse per day). Blood, liver, and cecal contents were collected immediately following euthanasia by CO2 asphyxiation. All samples were stored at −80 °C until analysis.Mouse experimental procedures were performed according to the National Institutes of Health (NIH) guidelines and approved by the Pennsylvania State University Institutional Animal Care and Use Committee . Four-week-old male C57BL/6J mice (22–25 g) were purchased from the Jackson Laboratory and housed at the Centralized Biological Laboratory of Pennsylvania State University . Mice . Aliquots were taken from the bacterial DNA extracts and all samples were brought to a concentration 10 ng/µL. Using 1 µL of each aliquoted sample and the Platinum SuperFi master mix (Invitrogen), amplification of the V4 (515F and 806R) region was completed with PCR (1 cycle at 98 °C (2 min); 25 cycles of 98 °C (10 s), 56.6 °C (20 s), 72 °C (15 s); and 1 cycle at 72 °C (5 min)). Amplicons were then checked on a 1X agarose gel and then submitted to the Pennsylvania State University Genomics Core Facility for 250 × 250 paired end Illumina Miseq sequencing. Blank samples and mock community standards  were analyzed with the test samples to check for contamination and check the error rate for the extraction and sequencing method, respectively. Raw data was returned and 16S rRNA gene amplicon analysis was completed using mothur software (version 1.39.5) and the SILVA database (2017 version) ,42. AfteThe raw 16S data were realigned with the GreenGenes (gg_13_5_99) database for a PICRUSt analysis . PICRUStg for 10 min. The combined supernatants were dried in a vacuum and reconstituted in 60 μL of 3% methanol. After centrifugation , 50 μL of supernatants were transferred to autosampler vials for LC-MS analysis. Metabolites were measured on a Dionex Ultimate 3000 quaternary HPLC system  coupled to an ExactiveTM Plus Orbitrap mass spectrometer (Thermo Fisher Scientific) with a Hydro-RP C18 column . LC-MS data were analyzed by an open-source software pipeline MS-DIAL [T test, p < 0.05 and a false discovery rate Q < 0.05.Cecal sample preparation for LC-MS and data analysis was performed as previously described . In brie MS-DIAL  for comp MS-DIAL ,27,28 foSimple Annotation of Metatranscriptomes by Sequence Analysis) pipeline [T test, p < 0.05 and a false discovery rate Q < 0.05.In total, 100 mg of cecal contents were used for RNA isolation using a 1:10 trizol/chloroform extraction. Total RNA was then measured and checked via an Agilent Bioanalyzer. The RiboZero isolation kit from Illumina was used to remove 16S and 23S rRNA from the samples. The remaining mRNA was checked again with the Agilent Bioanalyzer to ensure depletion of the 16S and 23S fractions. All mRNA was then submitted to the Pennsylvania State University Genomic Sequencing core for single-end RNAseq analysis on the Illumina Hiseq 150 bp). Unfiltered raw data was returned and analysis was completed with the SAMSA2 (version 2.0.0)  was used according to the manufacturer’s protocol. A standard curve was created with the provided endotoxin, diluted to 1, 0.5, 0.25, and 0.1 EU/mL. Serum samples were run in duplicate . Plates were analyzed for absorbance at 405 nm on a Synergy HTX multimode plate reader (BioTek).The PierceTM LAL Chromogenic Endotoxin Quantitation assay according to the manufacturer’s protocol.Then, 10 mg of cecal contents were first diluted in 10 mL of endotoxin-free water. A 500-µL aliquot was removed and sonicated with a probe sonicator. In total, 6 µL of each sonicated aliquot was then diluted in 1 mL of endotoxin-free water. The diluted sample was then used for the Piercen = 18) were added to a 96-well plate for QPCR analysis. QPCR was ran with the following conditions: 95 °C for 20 s; 95 °C for 0.01 s; 60 °C for 20 s; 95 °C for 15 s; 60 °C for 15 s; and 95 °C for 15 s, for 45 cycles. ΔΔCT values were obtained and a previously developed standard curve with the line of best fit equation (1) was used to determine CFU counts of the samples.An aliquot of extracted bacterial DNA was diluted to 1 ng/µL for each sample. Then, 3 µL of DEPC water, 1 µL of universal forward primer (AGAGTTTGATCCTGGCTCAG), 1 µL of universal reverse primer (CTGCTGCCTCCCGTAGGAGT), 6 µL of SYBR green QPCR master mix (BioRad), and 1 µL of each sample , zonula occludens 1 (Zo-1), and zonula occludens 2 (Zo-2) . RNA extracts were normalized to 500 ng/µL, and 2 µL of RNA was used to synthesize cDNA with qScript cDNA SuperMix (Quanta bio). Then, 1 µL of cDNA was mixed with Fast SYBR Green Master Mix (applied Biosystems) and appropriate primers for occludin (


After confirmation of PAH, animals received intravenous injection of 5.105 hMSCs or vehicle, followed by oral treatment with lodenafil carbonate (10 mg/kg/day) for 14 days. The ratio between pulmonary artery acceleration time and RV ejection time reduced from 0.42 ± 0.01 (control) to 0.24 ± 0.01 in the SuHx group, which was not altered by lodenafil alone but was recovered to 0.31 ± 0.01 when administered in association with hMSCs. RV afterload was confirmed in the SuHx group with an increased RV systolic pressure (mmHg) of 52.1 ± 8.8 normalized to 29.6 ± 2.2 after treatment with the association. Treatment with hMSCs + lodenafil reversed RV hypertrophy, fibrosis and interstitial cell infiltration in the SuHx group. Combined therapy of lodenafil and hMSCs may be a strategy for PAH treatment.Pulmonary arterial hypertension (PAH) is characterized by the remodeling of pulmonary arteries, with an increased pulmonary arterial pressure and right ventricle (RV) overload. This work investigated the benefit of the association of human umbilical cord mesenchymal stem cells (hMSCs) with lodenafil, a phosphodiesterase-5 inhibitor, in an animal model of PAH. Male Wistar rats were exposed to hypoxia (10% O Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary artery (PA) wall remodeling and hypertrophy , while pDrug classes currently approved for PAH treatment focus on promoting pulmonary artery relaxation , thus reOne promising alternative in this regard is the use of mesenchymal stem cells (MSCs), which could interfere with the production and release of paracrine factors with regenerative and immunomodulatory properties . In PAH The present work investigated the profile of the association of a PDE5 inhibitor, a drug class already in clinical use for PAH treatment, with hMSCs to identify a better and effective treatment than their use in monotherapy. Thus, the potential beneficial effect of the association lodenafil + hMSCs was evaluated in a hypoxia-induced PAH model in rats.®, Universidade Federal do Rio de Janeiro). The antibodies used were: 1. alpha smooth muscle actin  from Sigma-Aldrich ; 2. tumor necrosis factor alpha , c-fos (c. ab7963), p38 mitogen-activated protein kinase  and inducible nitric oxide synthase  from Abcam ; and 3. extracellular signal-regulated kinase 1/2  and phosphorylated extracellular signal-regulated kinase 1/2  from Cell Signaling Technology . Secondary antibodies goat anti-rabbit IgG (1706515) and goat anti-mouse IgG (1706516) horseradish peroxidase (HRP) conjugated secondary antibodies were obtained from Bio-Rad  and goat antimouse/anti-rabbit IgG F(ab’)2-HRP polymer conjugate (414191F) was purchased from Nichirei .Lodenafil carbonate was kindly donated by Cristália Produtos Químicos e Farmacêuticos Ltd.a.  and was dissolved in dimethylsulfoxide (DMSO) at a concentration of 10 mg/mL. SU5416 was synthetized at Laboratório de Avaliação e Síntese de Substâncias Bioativas  of the DNase I solution.Umbilical cord hMSCs were isolated and immunophenotyped as described by Alencar et al. . At the 2) in a ventilated acrylic chamber controlled by an oxycycler  for 21 days. Together, animals received weekly i.p. administration of SU5416 (20 mg/kg), a vascular endothelial growth factor (VEGF) receptor inhibitor . SU5416/hypoxia-induced PAH (SuHx) provides characteristics similar to the human disease [SuHx + vehicle (DMSO p.o.);SuHx + lodenafil (10 mg/kg/day p.o.);5 cells i.v.);SuHx + hMSCs (5.105 cells i.v.).SuHx + lodenafil (10 mg/kg/day p.o.) + hMSCs . To induce PAH, Wistar rats (180–250 g) were exposed to normobaric hypoxia (10% Onhibitor . Control disease . Doppler disease . After PhMSCs or the vehicle (DNase I in PBS 0.6 U/mL) were injected in the caudal vein of rats on a single dose after the period of 21 days in a hypoxia environment. Oral administration (gavage) of lodenafil carbonate or DMSO was once a day for 14 days. Echocardiography was repeated 12 h after the end of oral treatment followed by RV catheterization.Echocardiography evaluation was done by a unique operator, who was blinded to the experimental groups. Echocardiography was performed in animals under anesthesia by isoflurane   using a system equipped with a 25 MHz probe and a 15 mm focal length . Besides acquisition of Doppler pulmonary outflow, echocardiography was used to obtain RV and left ventricle (LV) parameters such as internal area, and wall thickness used B-mode and M-mode, respectively. Data were collected and analyzed by a blinded experimenter.Under anesthesia by ketamin  and xilazin , a pressure–volume catheter  connected to a pressure transducer  was introduced through the jugular vein in the rats. The RV systolic pressure (RVSP) was recorded on a computer for analysis using Lab Chart software . At the end of the experiment, animals were euthanized, and heart and lungs were removed.After removal, heart and lung fractions were either stored at −80 °C or fixed through immersion in formaldehyde 10% solution and embedded in paraffin. RV hypertrophy was evaluated through the determination of the ratio between RV weight and LV plus septum (S) weight (RV/VLV + S), Fulton’s index .2 1.2, KH2PO4 1.2, glucose 11.5, NaHCO3 15.5, CaCl2 1.2, bubbled with 95% O2/5% CO2 and maintained at 37 °C. After a 2 h equilibrium period of 1.5 g resting tension, preparations were exposed to increasing concentrations of phenylephrine (1 nM–10 μM). After the plateau of maximal contraction, the arteries were exposure to increasing concentrations of acetylcholine  to determine the vasodilatory activity and endothelial function. ACh-induced response curves were obtained using Lab Chart software .Isometric tension recording of PA reactivity was used to evaluate the endothelial function integrity . BrieflyProtein expression was measured as published elsewhere . BrieflyPulmonary arteriole muscularization was evaluated using lung slices (5 μm) rehydrated and exposed for 60 min to bovine serum albumin 5% in phosphate-buffered saline (PBS-BSA) solution. Then, tissues were incubated with primary antibody (1:500 anti-α-SMA in PBS-BSA 1%) for 2 h followed by exposure to the secondary antibody (2:3 Histofine Rat PO (Multi) in PBS). After washout with PBS, slices were stained and exposed to 3,3-diaminobenzidine for 5–10 min, counterstained with hematoxylin. Within each lung, 10–20 pulmonary arterioles with external diameter <50 μm were photographed with a digital camera  coupled to an Axiostar optical microscope  at 1000× magnification. Vessel medial wall area was expressed as the percentage of the wall portion positively stained with α-SMA relative to the total transversal area.Tissue fibrosis was evaluated through picrosirius red (PSR) staining. In pulmonary arterioles, 10–20 arterioles  per lung slice were photographed, under a 1000× magnification and perivascular collagen content was measured as the collagen area surrounding an arteriole normalized by its own transverse area. In the RV, 5–10 fields per section were photographed under 400× magnification and interstitial fibrosis was measured by obtaining the total collagen area per tissue area.In lungs stained with hematoxylin and eosin (HE), 10–20 pulmonary arterioles (diameter <50 μm) per lung slice were photographed under 1000× magnification and divided by the arteriole area for the perivascular cell count, an indicator of inflammatory infiltrate. The same technique was used in RV sections under 400× magnification.c-fos content in RV and lungs sections was evaluated using immunohistochemistry after incubation in peroxidase-conjugated secondary antibody for 2 h, followed by DAB staining, and was expressed by the percentage of cardiomyocytes with stained nuclei in fields under 1000× magnification. p38 MAPK, TNF-α and iNOS content evaluation in RV sections was also performed and protein content was expressed by percentage stained area of the total field area under 400× magnification.p < 0.05.For statistical analysis, the one-way ANOVA test was used followed by Tukey’s post hoc test using GraphPad Prism, version 6 . Differences were considered statistically significant if p < 0.01), indicating a reduction in the increased pulmonary pressure. RVSP of control rats was of 24.0 ± 3.1 mmHg . Therefore, when lodenafil was associated with hMSCs, it showed a further reduction to 15.8 ± 0.8 × 103 cells/μm2.Hypertrophy of pulmonary arterioles was detected in lungs from the SuHx group because the medial wall area was increased when marked with α-SMA using immunohistochemistry A. In con6 ± 0.03 B. PAH inells/μm2 B. In the3 cells/μm2, respectively  therapy was previously reported to reverse the alterations induced by PAH in mice [UC-hMSCs  .Lodenafil + hMSCs altered the expression of proteins involved in cellular proliferation and inflammation related to PAH, indicating the anti-proliferative and anti-inflammatory effects . FunctioMSCs release several paracrine mediators, which could interfere with the evolution of PAH ,31. ThesA downstream signaling molecule related to the proliferation phenotype in PAH is c-fos, whose expression is increased at pulmonary arterioles and is responsible for enhanced pulmonary artery smooth muscle cell proliferation in an animal model and in PAH patients . Since cERK1/2 is a member of the MAPK superfamily and a potent regulator of cell growth that can be activated by various extracellular stimuli, such as mitogens, growth factors and cytokines , and it p38 MAPK is another member of the MAPK superfamily that is upregulated in PAH, whose activation is correlated with fibroblast-to-myofibroblast differentiation by pathways dependent on TGF-β1 and TRPC6 , and incTNF-α is a pro-inflammatory mediator whose serum level is elevated in PAH patients . BesidesA receptors are detected in the RV of PAH patients [Endothelin-1 signaling promotes cardiac hypertrophy through c-fos-dependent pathways . Alteratpatients . An incrpatients , and conpatients , it is p2+ channel activity in RV cardiomyocytes, which is responsible for cardiac dysfunction [iNOS is associated with inflammatory cytokine effects in the heart leading to RV failure in monocrotaline-induced PAH animals . ERK1/2-function . LodenafCombination therapy of lodenafil and hMSCs reversed the functional, structural and molecular changes induced by SuHx-PAH through anti-inflammatory and anti-proliferative action, and may be an alternative therapeutic strategy for PAH treatment.