Dopamine release and phase-amplitude cross-frequency coupling (CFC) have independently been implicated in prefrontal cortex (PFC) functioning. To causally investigate whether dopamine release affects phase-amplitude comodulation between different frequencies in local field potentials (LFP) recorded from the medial PFC (mPFC) of behaving rats, we used RuBiDopa, a light-sensitive caged compound that releases the neurotransmitter dopamine when irradiated with visible light. LFP power did not change in any frequency band after the application of light-uncaged dopamine, but significantly strengthened phase-amplitude comodulation between delta and gamma oscillations. Saline did not exert significant changes, while injections of dopamine and RuBiDopa produced a slow increase in comodulation for several minutes after the injection. The results show that dopamine release in the medial PFC shifts phase-amplitude comodulation from theta-gamma to delta-gamma. Although being preliminary results due to the limitation of the low number of animals present in this study, our findings suggest that dopamine-mediated modification of the frequencies involved in comodulation could be a mechanism by which this neurotransmitter regulates functioning in mPFC. Here we set out to assess the functionality of RuBiDopa on LFP recorded from freely-behaving rats. Other important improvement that optical methods present over the exogenous injection of neurotransmitter is that the latter, due to diffusion limitations, does not correctly represent physiological conditions, while the rapid activation of receptors via optical methods yields activation timing that mimics the tonic (slow) or phasic (fast) release of neurotransmitters  plays a central role in the executive functions undertaken by the prefrontal cortex Cl)]PF6 was synthesized as previously described  was added to 20 mL of methanol/water solution (1:1) and refluxed under N2. Then a solution of trimethylphosphine in THF  was added by syringe. The reaction was followed by observation of UV–Visible (UV–Vis) spectra. In some cases, additional phosphine solution was added. Once the UV–Vis spectrum was stable, methanol and excess phosphine were removed by vacuum distillation. The resulting aqueous solution was filtered to remove any solids, and precipitated by the addition of excess of KPF6 at 0°C. The resulting dark orange solid was washed three times with cold water and dried. This precursor was solubilized in water by adding 100 mg of [Ru(bpy)2(PMe3)Cl)]PF6 to 3 mL acetone, and 3 mL of an aqueous suspension of 1 g of anionic resin Dowex-22 (chloride form). Before removing acetone by rotary evaporation, precursor was left in Dowex 22 for 10 min to allow PF6/Cl exchange. Then, 100 mg of dopamine.HCl were dissolved in the precursor complex solution under nitrogen atmosphere. After bubbling of N2 for 15 min, 2.5 equivalents (50 mg) of solid NaOH were added. The solution was heated under N2 during 16 h to prevent dopamine oxidation, cooled to 0°C and neutralized with 120 μL of acetic acid. In order to remove insoluble material, the solution was filtered and poured over excess of saturated solution of KPF6 to precipitate RuBiDopa. The product was purified by redissolving three times in an aqueous suspension of Dowex-Cl and further reprecipitation with KPF6. The redissolution/precipitation cycle was performed three times, and the aqueous solution obtained was lyophilized in dark, yielding a very hygroscopic orange powder. Synthesis process was performed in the dark to avoid photolysis of the complex.All reagents were obtained from Sigma-Aldrich and used without further purification. The precursor complex [Ru(bpy)It should be noted that it is not possible to perform a negative control injecting the caging component only, without DA, for this “caging component” does not exist as is. These Ruthenium complexes have 6 coordination positions and all 6 positions are occupied by a ligand in any stable complex. Therefore, one can replace other ligand for dopamine, but not leave it “empty.” As any replacement will change some chemical properties  would not be a good choice for a control.ad libitum. All animal care including housing, surgical, and recording procedures were approved by the Ethics Committee for Animal Use of the Federal University of Rio Grande do Norte (permit # 11/2015 CEUA-UFRN).Three male Wistar rats  obtained from our breeding colony were used for multiple neurophysiological recording sessions. Animals were kept under a 12-h light-dark cycle (lights on at 07:00) with food and water Animals were anesthetized with ketamine and xylazine and implanted with multiple 50 μm Teflon-coated tungsten microwires  partitioned in two 2 × 4 arrays with a 900 μm diameter cannula in the middle Figure .Animals were allowed for recovery for 1 week before recordings were performed. Experiments were carried out in the afternoon and animals were allowed to spontaneously traverse the sleep-wake cycle Figure . AnimalsThe multielectrode arrays were mounted with a cannula in the center, through which solutions were injected and a fiber optic was placed to uncage dopamine from RuBiDopa Figure . After ~All analyses were performed using customized code in MATLAB . For group analysis, all electrodes of the matrix were used , while for figures where data from one representative electrode is shown, we selected the channels with the highest values of comodulation.j is the normalized mean amplitude in bin j given byf is the amplitude series of the filtered signal (f denotes the center frequency). And finally, the MI is calculated asmax is obtained from the uniform distribution, in which pj = 1/N and then, Hmax = log N. Therefore, MI-values range between 0 and 1; due to the normalization, low entropy values (H) result in high MI-values. The MI is only computed for one frequency pair; a comodulation map, or comodulogram  in the final comodulogram . Time-frequency spectral decompositions were obtained by using the spectrogram function from the Signal Processing Toolbox, and power spectral densities (PSD) were calculated using the pwelch function from the same toolbox. Figure Filtering was performed using All periods were defined by spectral analysis combined with visual inspection of the video recordings. REM epochs were defined as periods of quiescence, REM sleep posture, and elevated theta power (theta/delta > 4), while SWS epochs were defined as periods of quiescence, SWS posture and low theta power (theta/delta < 4). Active waking periods were obtained by visual inspection of locomotion behavior and dominating theta oscillations, while quiet waking epochs were obtained as absence of exploratory behavior, clear signs of waking, and absence of theta oscillations.We characterized phase—amplitude coupling patterns of the LFP recorded in the mPFC during spontaneous behaviors and under the influence of dopamine uncaging.p < 0.05, Friedman's test). We also found evidence that gamma oscillations are reduced or even absent during REM sleep  in the mPFC. Figure 10 Hz in p Figure .Dopamine uncaging by light did not produce overt behavioral changes. Interestingly, after dopamine uncaging, the strength of the delta-gamma comodulation significantly increased when compared to the other conditions of the experiment  and 1 μM  and diffusion. The kinetics of these two processes compete and diffusion seems to have more influence in determining the net extracellular concentration of DA and thus the amount of DA that reaches DARs  , and impaired neuronal synchronization within cortical networks in the gamma frequency band has been proposed to contribute to several psychiatric disorders also characterized by cognitive deficits. In the PFC, it has been proposed that dopamine modulates working memory capacity (Seamans and Yang, The present data also constitute the first evidence of theta-gamma comodulation in the mPFC of freely behaving rats during active exploration, in agreement with recent results in mice (Zhang et al., Plastic changes related to DA input may also underlie delta-gamma comodulation observed after laser activation of RubiDopa or DA injection. DA fibers terminals arising from VTA innerve the same area as hippocampal inputs to PFC (Carr and Sesack, The low number of animals presented in this study is a highly significant limitation in the conclusions we can obtain from our results. Nevertheless, the effect observed after RuBiDopa uncaging is robust and we propose these findings as the preliminary stage from which further investigation can be founded. Future directions point to the replication of these results and additional experiments aiming to strengthen our conclusions and broaden our understanding about the role of DA in the modulation of cognitive processes in the prefrontal cortex.The study was designed by VA, RE, and SR. The chemical syntheses were performed by VA and RE. The experiments were performed by VA and AS. The analyses were performed by VA and RS. The figures were prepared by VA, RS, and SR. The manuscript was written by VA, RS, AT, and SR.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Despite substantial effort devoted in the history of supramolecular chemistry, synthetic supramolecular systems still lag behind biomolecular systems in terms of complexity and functionality. This is because biomolecular systems function in a multicomponent molecular network under out-of-equilibrium conditions. Here we report two-component supramolecular assemblies that are metastable and thus show time-dependent evolution. We found that the systems undergo either self-sorting or coassembly in time depending on the combination of components. Interestingly, this outcome, which had been previously achievable only under specific conditions, emerged from the two-component systems as a result of synergistic or reciprocal interplay between the coupled equilibria. We believe that this study sheds light on the similarity between synthetic and biomolecular systems and promotes better understanding of their intricate kinetic behaviors. It would be intriguing in terms of emergent behavior if the outcome generated from such a mixture of molecules is different from that obtained using individual molecules. In this way, the concept of molecular self-assembly will be expanded to facilitate ever more powerful means of designing new types of smart adaptive materials. To this end, a system under kinetic control offers many opportunities because it may give rise to diverse outcomes depending on the self-assembly pathways rather than the thermodynamic stability39. Research in this area has attracted increased attention and has recently been documented in several comprehensive reviews35.A key to enhancing the complexity of synthetic supramolecular systems, ultimately toward the level of biomolecular systems, is to establish a molecular network that consists of many different molecules. In a pool, molecules collide, interact and/or react with each other, and determine whether to self-sort or coassemble as if computing the best outcome1 and 5, (b) porphyrin 2, and (c) porphyrin 6. As discussed below, when two of these porphyrins are mixed, a metastable two-component supramolecular assembly is temporarily formed and then shows time-dependent evolution. Interestingly, the systems undergo either self-sorting or coassembly depending on the combination of components 39. A nanoparticle of 2 is in the thermodynamically stable state, whereas those of 1, 5, and 6 are in metastable states and change into thermodynamically more stable nanostructures (either nanofibers or nanosheets) over time. As demonstrated previously through seeded growth experiments, both nanofibers and nanosheets form in an autocatalytic manner. Absorption spectral measurements indicated that nanoparticle, nanofiber, and nanosheet structures consist of different porphyrin stacking modes, that is, J, H, and short-slipping J aggregates, respectively ) was cooled from 373 to 308 K, nanoparticles were spontaneously formed; that moment was then defined as ely Fig. 44–46.t = 0) outward, and each molecular system is briefly described below.When two of these porphyrins were mixed and treated likewise, coassembled J aggregate nanoparticles were formed, which after a lag time showed time-dependent evolution. Figure 38 The J aggregate of porphyrin 1 (and that of 5 as well) is in equilibrium with the monomeric porphyrin and is present as a metastable state for a few hours. Once the monomer nucleates the H aggregate, the J aggregate is consumed in elongation of the H aggregate. The transformation shows a sigmoidal kinetics characteristic of an autocatalytic process, and we define the time at which the transformation is 50% complete as t50 37 The bulky substituents in 2 prevent face-to-face stacking of porphyrin planes . In addition, 2 does not have a driving force for nanosheet formation . Therefore, the J aggregate of 2 is incapable of transformation, and thus is thermodynamically stable.(b)39 The metastable J aggregate of 6, as an on-pathway intermediate, rearranges into the short-slipping J aggregate and assembles into nanosheet structures along pathway B. This process was also found to be autocatalytic and is characterized by the t50 value . However, we found that strong agitation such as sonication facilitated nucleation of the H aggregate, and as a result, the pathway was switched. Now, as in the case of (a), the J aggregate of 6 acts as an off-pathway intermediate and is transformed into the H aggregate along pathway A. Thus, the J aggregate of 6 is capable of differentiation47.(c)37 A mixture of 1 and 2 forms a metastable two-component J aggregate, but after a lag time, the system undergoes self-sorting into the H aggregate of 1 and the J aggregate of 2. An increased proportion of 2 in the mixture impedes H aggregate nucleation of 1; this is how the t50 value for the self-sorting process is determined. Using this mechanism, we succeeded in programming the time-dependent evolution. Ghosh et al.14 and Yagai et al.15 have recently reported a similar time-dependent self-sorting process.(d)6 (c), we sought to investigate the two-component systems in which 6 is involved. We found that:Having witnessed the unique time-dependent self-sorting of the two-component system (d) and recently discovered the unprecedented differentiation behavior of 2/6 J aggregate undergoes self-sorting into the J aggregate of 2 and the H aggregate of 6.(e) The metastable two-component 5/6 J aggregate is transformed into a coassembled 5/6 H aggregate.(f) The metastable two-component 6, in its pure form, had previously been obtainable only by applying sonication. We assert that pathway complexity plays an important role in determining the final outcome. Although more intricate molecular networks have been reported to date, particularly in the field of systems chemistry20, the scheme shown in Fig. These new findings are intriguing considering that the H aggregate nanofiber of 37, we confirmed that porphyrins 1 and 2 coassembled into the J aggregate (nanoparticles) as long as the 2 content was less than 30 mol%. Thus, we investigated the time-dependent evolution of 2/6 mixtures under such conditions, that is, 10/90, 20/80, and 30/70 molar ratios of the 2/6 components. Cooling of a hot MCH solution of the 2/6 mixture produced the J aggregate  depended on the concentration of 2, which suggests that 2 was not involved in the transformation and self-sorted as the J aggregate . Thus, the two-component 2/6 J aggregates can be regarded as “mutants” having the sterically demanding moiety in 2 as a defect, and therefore as incapable of forming nanosheets. In the pool, sufficient “pure” J aggregate of 6 cannot statistically be accumulated to form a nanosheet, and instead, coexisting monomeric 6 reciprocally nucleates the H aggregate, thereby biasing the pathway to nanofibers. The fact that the t50 values became longer with increasing the proportion of 2 also excludes the possibility of the cross-catalytic process. Thus, 6 behaves similarly to 1 (and 5) in a two-component system with 2; in fact, the t50 values of system (e) were comparable with those of (d)37.Why did porphyrin 5 and 6  but the H aggregate (nanofibers), as evidenced by the absorption spectra  of the 5/6 system were independent of the mixing ratio, which suggests that all of the J aggregate was consumed  without transforming into the short-slipping J aggregate , H aggregate nucleation synergistically leads 6 to pathway A, which results in coassembled H aggregate nanofibers. On the other hand, in the 2/6 system (e), the presence of 2 prevents pathway B, so 6 is reciprocally guided to pathway A. In both the two-component systems, pathway B was shut down; we therefore assert that the outcome of the on-pathway intermediate is susceptible to changes in the network of molecular self-assembly. As illustrated here, unveiling the entire picture of pathway complexity is important for controlling the outcome of a molecular network. We note that the present system has some similarities with crystal polymorphism and amyloid fibrillation and may promote better understanding and hence control of such intricate phenomena at the molecular level. More practically, the concept of coupled equilibria should be useful to advance programmed supramolecular polymerization and its applications62.To conclude, it is worth reviewing the diagram shown in Fig. ion Fig. , and patSupplementaryInformation


For such a task, the production of these nanofibers by means of the electrospinning technique has been optimized. Their morphology and size have been characterized by optical microscopy and scanning electron microscopy (SEM). By selecting the optimal conditions to achieve the smallest and most uniform nanofibers, approximate diameters of up to 800–900 nm were obtained. It was also determined that the therapeutic agents that were used were encapsulated with high efficiency. The analysis of their stability over time by GC-MS showed no significant losses of the encapsulated compounds 15 days after their preparation, except in the case of methyl salicylate. Likewise, it was demonstrated that the therapeutic compounds that were encapsulated conserved, and even improved, their capacity to activate the transient receptor potential cation channel 1 (TRPV1) channel, which has been associated with the formation of psoriatic lesions.Psoriasis is a chronic autoimmune systemic disease with an approximate incidence of 2% worldwide; it is commonly characterized by squamous lesions on the skin that present the typical pain, stinging, and bleeding associated with an inflammatory response. In this work, poly(methyl vinyl ether- Psoriasis is a chronic immune-mediated disease that affects 2% of the population, comprising different clinical manifestations, which are mainly characterized by skin disorders such as erythematous papules or scaly teardrop-like lesions associated with the characteristic pain, stinging, and even bleeding of an inflammatory process. The most affected areas are usually the elbows, knees, scalp, and lower back ,2.The immune system plays a crucial role in the pathogenesis of the disease, and particularly, the deregulation of the crosstalk between the innate and adaptive immune system within the interleukin-23 (IL23)/T helper cell 17 (Th17) axis . Thus, tThe main palliative agents for the dermal symptomatology of the disease are keratolytics, to reduce the generation of scales, and analgesics, to relieve the painful and itching sensation. Among the keratolytics, salicylic acid stands out, which acts by lowering the pH of the stratum corneum and thus preventing the adhesion of keratinocytes . In turn2+) that has been involved in the transmission and modulation of pain when triggered by different physical and chemical stimuli such as temperatures above 43 °C, acidic pH, capsaicin, and some endogenous mediators of inflammation [Capsicum, and some of its derivatives act as analgesic agents by producing TRPV1 desensitization [In terms of analgesic therapy, one of the most common targets in topical applications is the transient receptor potential cation channel 1 (TRPV1), commonly called the capsaicin receptor. This receptor is mainly found in the nociceptive neurons of the peripheral nervous system, although they have also been described in other non-neuronal tissues, including skin cells. TRPV1 is a non-selective tetrameric cation channel with high permeability to calcium  (PMVEMA-ES) as the polymeric material. Such a biodegradable, biocompatible, bioadhesive, and low-toxic polymer is already commercialized, and used in other biomedical applications, and has already shown its suitability for the creation of these nanostructured systems with the appropriate release characteristics [In this work, based on the progress made in our previous study , the proeristics ,28,29,30eristics ,32, in oeristics ,33,34. Talt-maleic acid) monoethyl ester (PMVEMA-ES) (Mw: ~130 kg/mol) was supplied in a 50% w/w solution in ethanol .The copolymer of poly, methanol , sulfuric acid, and anhydrous dimethyl sulfoxide (DMSO) (Sigma-Aldrich).The reagents used were salicylic acid, capsaicin, ruthenium red (RR) (Sigma-Aldrich), magnesium sulfate anhydrous , sodium hydroxide , and ferric chloride (Sigma-Aldrich). Although it is considered in the text as a unique therapeutic agent, according to the supplier’s analysis certificate, the purity of the commercial capsaicin used in this work was 61.1%, and it contained 31.2% of dihydrocapsaicin, which is a derivative with very similar chemical and biological properties to capsaicin.et al. (2016) [Methyl salicylate was synthesized from the esterification of salicylic acid in a solution of methanol and sulfuric acid by following the method proposed by Carrillo-Arcos . (2016) . Brieflyet al. (2017) [w/w of PMVE/MA-ES in ethanol was selected. As for the active agents, a combination of 1.5% salicylic acid, 1% capsaicin (2:1 together with dihydrocapsaicin), and 1% methyl salicylate, all w/w with respect to the polymer weight, was used. The preparation of nanofibers was performed by means of the methodology described before by Mira . (2017) . After oThe electrospinning process was performed on a device that included a two-mL Discardit II syringe  through which the polymer solution was introduced, and from which it was pumped through a blunt-end stainless steel hypodermic needle 316 of 20 Gauge  (Sigma-Aldrich) at a sustained flow controlled by a KDS 100 infusion pump . The needle and the aluminum foil collector, faced vertically, are connected to a Series FC high voltage source , which provides the voltage responsible for the generation of the jet to be deposited on the collector located at a settled distance. Any material that is intended to be covered with a mat of electrospun nanofibers, as for example in this work, the adhesive dressings, would be placed on the aluminum collector. After the optimization of the operational parameters (see corresponding section for further details), values were selected for the elaboration of the batches of nanofibers for the following experiments. For the observation in optical microscopy, the nanofibers were electropun on a microscope slide  arranged on top of the aluminum collector. The inverted fluorescence optical microscope that was used was a Microsystems DMI3000B  equipped with a Leica EL6000 compact light source and a Leica DFC 3000G digital camera. The images were taken with a 63× objective in phase contrast, and the image processing was done manually using the program Leica Application Suite AF 6000 Module Systems. By this methodology, an initial screening, and then a more exhaustive screening, were carried out. After the optimization of the electrospinning parameters, selected nanofiber samples were also analyzed by scanning electron microscopy (SEM), without metal coating, in a JSM-6360 LV device . In both cases, from the images obtained for each sample, a total of 100 measurements of nanofiber diameters were taken and analyzed by using the Image J software .The amount of the therapeutic agents contained in the nanofibers, which are necessary for analyzing the encapsulation efficiency and the stability of the encapsulation over time, was determined by means of a gas chromatograph coupled to a mass spectrometer, in particular, a GCMS-QP2010 SE equipment with quadrupole detector supplemented with a thermal TD-20 adsorption attachment and an AOC-20i/s automatic sample injector . The employed GC column was an Agilent J&W capillary HP-5MS UI  .The procedure of this analytical methodology is based on a previous study . BrieflyR2 coefficients greater than 0.99 in all of the cases  were used. The analyzed samples corresponded to two independent productions of nanofibers loaded with the three therapeutic agents as described above. Each sample of nanofibers was analyzed when just prepared and at 5 and 15 days post-production. These samples were stored in a drawer, uncovered, protected from light, and at room temperature. Immediately prior to their analysis, the samples were dissolved in dichloromethane, filtered with 0.2-μm nylon membranes  and injected at a concentration of 10 mg/mL in vitro cell assays, human embryonic kidney cells stably expressing TRPV1 (HEK293-VR1) were used. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% v/v fetal bovine serum (FBS) (Sigma-Aldrich) and 50 μg/mL gentamicin . Cells were cultured in 25-cm2 flasks at 37 °C in a humidified atmosphere with 5% CO2, and for their harvest they were detached with 0.25% trypsin-EDTA solution. For the assays, cells were seeded in opaque 96-well plates with transparent bottoms  at a cell density of 40,000 cells three days before treatment.For 2+ when activated, a Ca2+ fluorescence probe has been used to indirectly quantify such activation, which might be induced by the experimental treatments. Thus, Ca2+ fluorography assays were performed by using the probe Fluo-4 NW  supplemented with 2.5 mM of probenecid, which improves the permeation of the probe in the cells and inhibits losses of fluorescence by avoiding its exit to the cellular exterior, as was recently reported by Serafini et al. (2018) [Since the TRPV1 channel allows the non-selective passage of cations such as Ca. (2018) .Briefly, in cell plates prepared as described in the previous section, the culture medium was removed, and 100 μL/well of probe solution was added and incubated for 30 min at 37 °C followed by 30 min at 30 °C, always in the dark. Then, the fluorescence measurements were taken (excitation at 485 nm and emission at 535 nm) with a POLARstar Omega plate reader  for 20 cycles (each one corresponding to a period of 156 s) and at a constant internal temperature of 30 °C. After the third cycle, one μL/well of each formulation that had been previously dissolved in DMSO at the corresponding concentration was added manually in order to reach the expected treatment doses in the well. In this sense, nanofibers were dissolved at 50 mg/mL in order to treat cells with 54 μM, 33 μM, and 10 μM of salicylic acid, methyl salicylate, and capsaicin, respectively. A solvent control (DMSO), capsaicin as agonist compound at 10 μM , and RR in vivo study was carried out with three healthy human volunteers to evaluate, when administered topically, the stability time of the experimental nanofibers, as well as to determine the functionality of the encapsulated TRPV1 agonists by analyzing the responsiveness to them. Each volunteer was given an adhesive dressing with electrospun nanofibers on each arm on the skin surface of the deltoid. This test was blind, since for each volunteer, one of the dressings contained empty nanofibers, and the other contained drug-loaded ones, but they were assigned randomly to each of their arms. Each dressing contained approximately 25 mg of nanofibers distributed over a surface area of 20 cm2. The duration of the treatment was eight hours. The surface of the dressing was kept protected from light throughout the full period. Every two hours, photographs were taken of each dressing to determine the state of the nanofibers and treated skin. At the end of the trial, each volunteer was asked which dressing they thought contained the TRPV1 agonists. This study was approved by the Project Evaluation Board of Miguel Hernández University .An initial Data were analyzed by either GraphPad Prism v6 and Microsoft Excel software. Both of them were used for creating the graphs. Statistical analysis was performed with GraphPad Prism v6 specifically. Applied statistical methods are stated together with the analyzed data in the Results section.w/w), pumping flow (0.25–1.35 mL/h), voltage (6–17.5 kV), and distance between the needle and collector (8–30 cm). Initially, we proceeded to the optimization of the morphology and size of the nanofibers without the encapsulated agent. For this, the operational values of the electrospinning procedure were modulated within a range that had been estimated from our previous study . They inw/w of PMVE/MA-ES . TherefSo far, the analysis of the nanofibers had been done by optical microscopy; however, to analyze the morphology in more detail and with greater precision, the size of the optimized nanofibers was observed by SEM. The measurements obtained by electron microscopy are equivalent to those obtained by optical microscopy: 808 ± 31 nm for empty nanofibers, and 875 ± 49 nm for those with encapsulated therapeutic content . The low amount of encapsulated drug that was used in this work  does not explain the increase in size that was observed in the loaded nanofibers, even more considering that loading percentages are usually much higher for molecules that have a similar complexity, which even reduces nanofiber diameters ,41. NeitTo quantify the amount of encapsulated therapeutic agent, they were separated by gas chromatography, and then identified with mass spectrometry. In the calibration process, the elution times were precisely determined for the four compounds: methyl salicylate, 12.5 min; salicylic acid, 14.0 min; capsaicin, 29.0 min; and dihydrocapsaicin, 29.3 min . Such value was used as a reference for calculating the encapsulated content over time  in all cases when measuring right after the production of nanofibers  and even 45% in the case of the Reflex gel. In the case of capsaicin, it has been used at 1% w/w, as in the present study, with respect to the base polymer in nanofibers and nanoparticles for topical use in psoriasis [Qutenza Astellas, which is used to treat neuropathic pain.Regarding the concentration of each therapeutic agent proposed, salicylic acid is usually used at concentrations of 0.5–60% soriasis . The cone creams . The usee creams . The consoriasis ,54. In ain vitro skin permeability studies performed in our previous work to evaluate the potential of this polymeric family for topical applications [One of the most surprising results was the loss of product observed for methyl salicylate, which seems to be due to the increase in surface area with respect to the volume that occurs in one-dimensional materials when they are nanostructured, which increases the evaporation of the encapsulated substances . This efications , it was The activity of the TRPV1 agonist compounds encapsulated in the experimental nanofibers , alone or in combination with the other two agents , was assessed by treating HEK293-VR1 cells. These results were represented in percentages relative to the highest value obtained for the activation induced by capsaicin at 10 μM and using the lowest one after inoculating RR as the baseline.2+ after being inoculated in cycle three. The graph also shows that TRPV1 is highly activated right after the inoculation of each agent and remains at high levels, which even increase in the case of capsaicin. The specificity of the system is checked by the blocking of the channel with RR, which is even able to desensitize it to a subsequent stimulation with capsaicin (cycle 10). Firstly, in in vitro cell assays (p = 0.0011), meaning that the encapsulation process did not affect the chemical properties of the encapsulated agents. Some activation of TRPV1 was also observed when treating with empty nanofibers.For the comparison of the activity of the agonist compounds on TRPV1 when they are encapsulated in nanofibers, alone or together with the other therapeutic agents, the same above-described procedure was followed, but including experimental nanofibers that were previously dissolved in DMSO. In these l assays , althougThe combination of different compounds within the same formulation generally pursues a synergistic effect that could allow the reduction of the concentration of each one, and thus their potential adverse effects; however, other benefits from such an approach might be the combination of other functional properties such as, in this case, the ones introduced previously, which are also related to treating psoriasis and other factors such as their antimicrobial activities ,57.Finally, but also related to the assessment of the ability of the developed nanofibers to activate TRPV1, it has been possible to successfully electrospin the experimental nanofibers on adhesive dressings for topical application and preliminarily check their gradual degradation by visual observation, as well as their content release by assessing the typical reddening reaction of the capsaicin in the skin . Both efw/w all together with respect to the amount of polymer), with an encapsulation efficiency of approximately 100%. The stability studies of the content encapsulated over time (up to 15 days after its production) showed significant losses especially of methyl salicylate, which were probably due to its higher volatility. In vitro studies revealed that the TRPV1 agonist compounds maintained their channel-activating capacity at the expected levels after being encapsulated. Finally, it was found that the proposed nanofibers are suitable for the creation of skin adhesive dressings and allow the release of the encapsulated compounds by the disintegration of the nanostructure. In this study, the effect of those compounds with the capacity to activate TRPV1 was checked in vivo.In this work, it has been possible to optimize the electrospinning parameters to create PMVE/MA-ES nanofibers loaded with a combination of three compounds with therapeutic activity to alleviate the dermal symptoms of psoriasis (salicylic acid, methyl salicylate, and capsaicin at a concentration of 3.5%


The time-dependent (30 min - day 84) plasma profile of PT320, a sustained-release (SR)-Exenatide formulation under clinical development for treatment of neurodegenerative disorders, was evaluated in nonhuman primates after a single subcutaneous dose and was compared to Bydureon. Exenatide release from PT320 exhibited a triphasic pharmacokinetic profile. An initial peak occurred at 3 hr post-administration, a secondary peak at 5 days, and achievement of Exenatide steady-state plasma levels from day 10–28. Systemic exposure increased across PT320 doses, and Exenatide levels were maintained above the therapeutic threshold prior to achieving a steady-state. In contrast, Exenatide release from Bydureon exhibited a biphasic profile, with an initial plasma peak at 3 hr, followed by a rapid decline to a sub-therapeutic concentration, and a gradual elevation to provide a steady-state from day 35–49. Exenatide total exposure, evaluated from the area under the time-dependent Exenatide concentration curve, was similar for equivalent doses of PT320 and Bydureon. The former, however, reached and maintained steady-state plasma Exenatide levels more rapidly, without dipping to a sub-therapeutic concentration. Both SR-Exenatide formulations proved well-tolerated and, following a well-regulated initial release burst, generated steady-state plasma levels of Exenatide, but with PT320 producing continuous therapeutic Exenatide levels and more rapidly reaching a steady-state. Further to its well-characterized insulinotropic function and other actions on glucose homeostasis, produced by suppressing glucagon secretion from pancreatic α-cells, slowing gastric emptying and inducing satiety to reduce food intake and body weight, GLP-1 confers glucose sensitivity to glucose-resistant β-cells and provides trophic support by stimulating pancreatic β-cell proliferation and neogenesis, and by reducing β-cell apoptosis4. As the glucose homeostasis actions and enhancement of insulin signaling provided by GLP-1 are largely preserved in type 2 diabetes mellitus (T2DM), agents that activate the GLP-1 receptor (GLP-1R), a class B G protein–coupled receptor (GPCR) that mediates the action of GLP-1, have been developed for the treatment of T2DM; they appear to be well-tolerated, efficacious and widely used9. Furthermore, as the GLP-1R has been found expressed across a wide variety of other organs, such as on neurons within the brain and peripheral nervous system and within all four chambers of the heart, GLP-1R agonists are currently being evaluated across an increasing number of other diseases15.The incretin glucagon-like peptide 1 (GLP-1) is a gastrointestinal peptide hormone and proteolytic product of proglucagon. It is predominantly generated within and released by intestinal enteroendocrine L cells to potentiate glucose-dependent insulin secretion by pancreatic β-cells following food ingestion1/2 1.5 min) within the circulation following either its physiological release or direct administration, with proteolysis, principally by DPP-4 and renal clearance having major roles. DPP-4 cleaves the amino-terminal dipeptide from GLP-1 to reduce its activity17. The identification of this led to the strategies for (i) modifying the peptide to lower proteolysis/renal clearance to generate longer-acting incretin mimetics and (ii) to elevate endogenous GLP-1 levels by using DPP-4 inhibitors. In relation to the former, an increasing number of GLP-1R agonists have been developed for clinical use and, as trials and experience with them grow, it will become clearer whether one has advantages over another within specific clinical settings. A practical way to categorize clinically available GLP-1R agonists is based on their duration of action. Centered on their pharmacokinetic/ pharmacodynamic profile, they can be divided into short-acting (Exenatide (Byetta): T1/2 2.4 hr18, and lixisenatide T1/2 3 hr19: administered twice and once daily, respectively) and long-acting GLP-1R agonists (Exenatide long-acting release (LAR (Bydureon)), liraglutide T1/2 13 hr, albiglutide T1/2 6–7 days, and dulaglutide T1/2 4 days21: administered from once daily to weekly  and its extended release formulation, Exenatide as Bydureon, was approved in 2012 for once weekly dosing. Both are well-tolerated and remain widely used21. In contrast to liraglutide that reversibly binds to serum albumin, albiglutide that is the fusion product of two modified GLP-1 molecules with albumin, and dulaglutide that comprises two modified GLP-1 molecules joined by a linker to a modified human immunoglobulin heavy chain – each tailored to protect against DPP-4 activity and renal clearance, Exenatide as Bydureon is the same 39 amino acid peptide as used in Byetta but is encapsulated in poly(lactic-co-glycolic acid) (PLGA) microspheres to enable its sustained release from the subcutaneous reservoir generated at the injection site24. Modifications in how Exenatide is formulated into PLGA can transform the release kinetics and the resulting pharmacokinetics of the drug26. In the current study, we evaluated a new sustained release (SR)-Exenatide formulation, PT320, with a longer (2 week) duration dosing profile that is under clinical development by Peptron Inc.  for the treatment of neurodegenerative disorders29. PT320 has similar properties to PT302, formerly developed by Peptron for the treatment of T2DM30, and is manufactured utilizing an ultrasonic spray drying process, termed SmartDepot™31, to generate microspheres that contain 2% Exenatide. These microspheres likewise comprise the polymer, PLGA. Notably, they are of small uniform size (20 μm diameter). This allows subcutaneous administration using a smaller needle size  and, importantly, supports the rapid achievement of steady-state Exenatide levels. Additionally, their coating with L-lysine provides control of the initial burst of Exenatide released following PT320 subcutaneous administration. In the present study, time-dependent levels of Exenatide were evaluated in nonhuman primates following the subcutaneous administration of matched Exenatide doses within PT320 and Bydureon in order to compare their pharmacokinetic profiles.Exenatide was the first approved GLP-1R agonist for T2DM as a twice daily medication (Exenatide administration in the form of both PT320 0.44 and 1.1 mg/kg) and Bydureon (1.1 mg/kg) proved to be well-tolerated at the doses evaluated, albeit hard nodules  were evident within the subcutaneous injection site across groups . For one animal in the PT320 1.1 mg/kg group (Monkey #12), the nodule broke the skin surface and resulted in drug loss (occurring at approximately weeks 3 and 4), and hence this animal was removed from the pharmacokinetic analysis. The mean body weight of rhesus monkeys is shown in Fig. .1 mg/kg Itrough) between 60 and 72 hr. For animals administered either dose of PT320, a secondary peak was consistently evident at 5 days, and approximately steady-state Exenatide plasma levels were then achieved from days 10 to 28 post administration . In contrast, following Bydureon administration, plasma levels of Exenatide gradually and time-dependently increased to achieve an approximate steady-state concentration between day 35 to 49 (5 to 7 weeks) post injection; rapidly declining at week 8 and being minimal thereafter.An initial release burst of Exenatide was evident in plasma as early as 30 min after administration of either dose of PT320 or of Bydureon. Illustrated in Fig. 33. Notably, and shown in Table ipeak and Tipeak, following subcutaneous administration is remarkably similar between the matched 1.1 mg/kg doses of PT320 (1740.2 ± 86.1 pg/ml at 3 hr) and Bydureon (1960 ± 167.8 pg/ml at 3 hr) (p = 0.35). Likewise, the maximal concentrations, Cmax, achieved by these different formulations are also comparable; 1535.6 ± 167.7 and 1617.4 ± 272.5 pg/ml (p = 0.83), respectively. However, the time to achieve these, Tmax, was significantly shorter for PT320 compared to Bydureon  vs. 966 ± 80.4 h , p = 0.006), and is reflected in the different time-dependent AUC values , the lowermost Exenatide plasma concentration (CItrough) maintained by Bydureon was significantly less (8.6 ± 3.2 pg/ml at 24 hr) than that achieved by PT320  , TItrough: 60 ± 12 hr vs. 72 ± 0.0 hr (p = 0.4); Tmax: 438 ± 66 hr (18.25 days) vs. 416 ± 88 hr (17.3 days) (p = 0.85), respectively), the concentrations associated with these demonstrated dose-dependence (Cipeak: 924.5 ± 62.9 vs. 1740.2 ± 86.1 pg/ml (p = 0.0005); CItrough: 72.1 ± 10.9 vs. 137.7 ± 21.7 pg/ml (p = 0.03); Cmax: 1227.7 ± 251.3 vs. 1535.6 ± 167.7 pg/ml (p = 0.39), respectively).Also, of note is the similarity in the shape of the time-dependent Exenatide concentration curves generated by the two evaluated doses of PT320 (0.44 and 1.1 mg/kg) in nonhuman primates  to provide an initial evaluation of dose-dependence. Additionally, we compared the higher PT320 dose to a matched single dose of Bydureon (1.1 mg/kg) to evaluate the equivalences of these two different SR Exenatide formulations. Systemic Exenatide exposure was measured periodically for 84 days (2016 hr) following a single subcutaneous dose of these formulations. These doses were based on a prior Bydureon study in nonhuman primates (Cynomolgus monkeys) in which single doses of 0.11, 0.44 and 1.1 mg/kg were evaluated from 0.5 hr to 71 days, and proved to be well-tolerated35. The minimally effective concentration of Exenatide in humans has been reported as approximately 50 pg/ml, as this is the level required to reduce fasting plasma glucose levels36. In using 50 pg/ml as a ‘cutoff limit’, the PT320 0.44 mg/kg administration provided a therapeutic level of dosing for up to 31.5 days (756 ± 84 hr), the PT320 1.1 mg/kg dose for up to 44 days (1064 ± 112 hr), and the Bydureon 1.1 mg/kg dose for 52 days (1260 ± 48.5 hr) in nonhuman primates . These results are in accord with data from human studies evaluating the pharmacokinetics of Exenatide release from PT302 in which concentration-dependent release was measured for up to 55 days following a single subcutaneous dose of PT30230, and for some 60 days (8.5 weeks) following Bydureon37 and, additionally, are consistent with weekly to biweekly dosing38.The goal of the current study was to evaluate the pharmacokinetic properties of a single-dose of PT320 in healthy nonhuman primates. PT320 is a form of PT3028 as well as for other disorders14, Exenatide is routinely administered chronically; over months to years duration. This would involve once weekly dosing of Bydureon9 and either once weekly or once every other week dosing for PT32030. Albeit not the focus of our single dose study, such multiple dosing would be expected to impact plasma Exenatide levels achieved at 7 and 14 days onwards, after Bydureon and PT320, respectively, and to maintain steady-state levels over a far longer duration. Importantly, Exenatide plasma levels associated with multiple dosing of either Bydureon or PT320 would be similar to that achieved with a single dose prior to 7 and 14 days, respectively, as only a single dose would have been administered at these times.In the clinical setting for the treatment of T2DM30. This first phase is caused by the ‘burst release’ of Exenatide occurring from the PLGA nanospheres in which the drug is encapsulated. This predominantly derives from Exenatide on or close to the surface that enters the systemic circulation on initial nanosphere hydration prior to the onset of polymer erosion-associated Exenatide release39. An initial peak in plasma Exenatide was likewise evident following Bydureon administration to nonhuman primates at 3 hr, in line with human studies in which a peak at 2.1 to 5.1 hr was noted37. Our study indicates that the concentration of the initial burst release in nonhuman primates appeared to be well-regulated across both Exenatide SR formulations, and was dose-dependent, with the CIpeak value approaching that of the Cmax value ; and the variance around the CIpeak value is relatively small . This initial burst peak was relatively short in duration, with Exenatide plasma concentrations declining rapidly by 24 hr to provide a time-dependent concentration of less than 3% of the total exposure to Exenatide (AUC0-24 hr ÷ AUC0-2016 hr). This is in accord with studies of PT320 in humans in which the value of CIpeak was likewise comparable to CMax, and the initial Exenatide burst represented only a small fraction of the total drug exposure 22.A consistent initial peak in plasma Exenatide was noted following PT320 administration to nonhuman primates within 1.75 to 3 hr, which is comparable to the characteristic peak seen in humans that occurs within 4 hr of dosingItrough: 8.6 ± 3.2 pg/ml) at 24 hr across all (4/4) nonhuman primates and persisting at a sub-therapeutic level in 3/4 animals at 72 hr .Also evident from our time-dependent analysis of plasma Exenatide concentrations generated by PT320 or Bydureon in nonhuman primates Fig.  is the s hr Fig. . In contAve and CMax values was in the order of 2-fold . Bydureon briefly declined to sub-therapeutic levels following the initial burst phase, and then gradually increased over time  to achieve the CMax was shorter for PT320, which is reflected in the AUC values , with plasma Exenatide levels never falling below the minimal therapeutic concentration of 50 pg/mlime Fig. . Notablees Table . Also evin situ matrix drug reservoir. Continuous diffusion from this matrix results in the secondary release phase of Exenatide into plasma that, followed by the hydrolysis and erosion of the PLGA polymer, results in the steady-state Exenatide third release stage39. The PLGA polymer hydrolysis products, lactic and glycolic acids, are ultimately eliminated as carbon dioxide and water23. Whereas PLGA is a biodegradable and biocompatible medical polymer with a well-established safety profile as an excipient in a wide variety of controlled release drugs40, the appearance of subcutaneous injection site reactions for Bydureon is reported as 17.1% from clinical data presented within the US approved prescribing information41. When present, these reactions may occur after the first dose (70%), and cause local skin discoloration (70%), pain or discomfort (56%), pruritus (48%), warmth (33%), and swelling (30%) at the injection site. Most cases reported nodules characterized as hard, subcutaneous, lumps, masses, or indurations41, with a minority (22%) of cases reporting a concurrent abscess. The occurrence of injection site induration and pruritus was also noted in T2DM subjects administered PT320, and is described to be dose-dependent and to naturally resolve30. In our nonhuman primate study, such Bydureon and PT320 associated nodules were evident across all animals (12 of 12) with one demonstrating an abscess (1 of 12), and all self-resolved. In contrast, Bydureon in humans is routinely administered as a dose of 2 mg in a 0.65 ml injection volume (or 0.85 ml for Bydureon BCISE)22, and hence the doses and corresponding injection volumes are substantially larger in our nonhuman primate study , and likely resulted in a higher injection reaction site incidence and unusual loss of drug in nonhuman primate #12.Following the initial burst release phase of Exenatide from the PLGA microsphere surface of either PT320 or Bydureon, the microspheres hydrate, adhere to one another, and create an 43. Notable across both PT320 doses and Bydureon administration in our nonhuman primate study is the rapid decline in plasma Exenatide levels at the end of steady-state, coinciding with the exhaustion of the drug reservoir. This is in accord with a prior pharmacokinetic study of subcutaneous immediate release Exenatide in rhesus monkeys in which the terminal disappearance half-life from plasma ranged from 0.78 to 1.94 hr.44. Studies across animal species and humans indicate that Exenatide, whether administered by immediate or sustained release, is predominately eliminated by the kidney via glomerular filtration with subsequent enzymatic degradation45. Studies in nephrectomized rats demonstrated that Exenatide slowly disappeared from plasma signifying the occurrence of a non-renal clearance component46. This may involve target-mediated drug disposition47, encompassing the binding of Exenatide to the GLP-1R, and the formation of Exenatide-GLP-1R complexes that either dissociate or become internalized and targeted for receptor-mediated endocytosis and degradation48.Our study was specifically designed to follow time-dependent Exenatide levels achieved by the sustained release formulations PT320 and Bydureon, rather than to evaluate the Exenatide elimination half-life from plasma – which is generally unchanged when the same drug entity is administered either by immediate or sustained release preparation37 as well as in preclinical studies31, we evaluated whether such an occurrence transpired in our nonhuman primate study by evaluating plasma samples at 6 and 12 weeks. Two of 12 animals (16.7%) demonstrated a positive antibody titer. Interestingly, these two  were associated with lower values of plasma Exenatide  less commonly occur  and may diminish Exenatide efficacy37. Such antibodies do not appear to cross-react with human GLP-1 or glucagon, or influence the safety profile of Exenatide formulations37.In the light of reports of anti-Exenatide antibody development in human studies focused on Exenatide use in T2DMIpeak, CMax and CAve were equivalent, importantly the time to achieve CMax (TMax) and the steady-state occurred significantly earlier for PT320. Furthermore, the dramatic decline in plasma Exenatide levels that followed the CIpeak and resulted in the CItrough was attenuated in the PT320 formulation and, unlike Bydureon, continuously maintained therapeutic drug levels prior to reaching steady-state delivery. Evaluation of a lower PT320 dose (0.44 mg/kg) in nonhuman primates demonstrated dose-dependence in relation to CIpeak, CItrough, CMax and CAve values, with maintenance of the times to achieve these and the duration of the steady-state. Notably, our CMax and AUC (0–2160 hr) values for Bydureon 1.1 mg/kg are in accord with prior preclinical nonhuman primate data that supported the agent’s approval for clinical use (see: CMax and AUC (0–1704 hr) in35). For treatment of disorders involving the use of an SR-Exenatide formulation in which the rapid generation and long-term maintenance of therapeutic plasma and target levels is desired, as could be considered in the experimental treatment of mild and moderate head injury53 or acute ischemic injury57 , the pharmacokinetic characteristics of PT320 may prove particularly important; associated with the rapid achievement and maintenance of therapeutic Exenatide levels in plasma. For chronic disorders, such as T2DM and PD in which Exenatide has demonstrated value58, both sustained release formulations would appear to be valuable to achieve and maintain long-term steady-state drug levels through the dosing of Bydureon once weekly and PT320 either once every two weeks or once weekly.In summary, our evaluation of a matched dose of the SR-Exenatide formulations PT320 and Bydureon (1.1 mg/kg) in nonhuman primates demonstrated a similar, well-regulated, initial burst release of Exenatide into plasma within 3 hr. This was followed by a lag phase and a secondary and tertiary release stage of Exenatide to provide reasonably steady-state levels in plasma. Whereas the plasma levels of Exenatide associated with C35. Animals included 12 male (4–11 years old) rhesus macaques (Macaca mulatta) randomized into three treatment groups (n = 4). Monkeys were maintained at the National Institutes of Health Animal Center  and housed in standard primate caging with a controlled temperature and humidity and a 12-hr light cycle. Commercially prepared monkey chow was distributed twice per day along with daily food enrichment, and water was available ad libitum. Monkeys were observed daily for food consumption and overall well-being. Animal husbandry and all experimental procedures complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were conducted under approved protocols by the National Institute on Aging Intramural Research Program Animal Care and Use Committee.Nonhuman primates were used in this study to provide a closer translational link in relation to evaluating a new drug formulation destined for use in humans, as compared to studies in ratsAt each procedure, monkeys were restrained with ketamine , which was supplemented with partial doses of ketamine, as needed, to maintain anesthesia. Following an initial baseline blood draw to obtain background signal levels for Exenatide ELISA evaluations, one of three pharmaceutical grade treatments was administered subcutaneously into the abdomen  within each Exentatide sustained release formulation – to ensure matching of the Exenatide in PT320 and Bydureon . The treatment groups included (i) PT320 0.44 mg/kg , (ii) PT320 1.1 mg/kg , and (iii) Bydureon 1.1 mg/kg . A 4.0 ml blood sample was collected into an EDTA vacuum tube at 0.5, 3, and 24 hr, at days 3, 5, 7, and 10, and then once per week throughout the 12-week study . Blood samples were immediately placed on wet ice, centrifuged within 30 min  and, following plasma removal, separate aliquots were frozen and stored at −80 °C.Exenatide levels were quantified by using the Peptron Exendin-4 EIA Kit . Each sample was evaluated in duplicate at a volume of 50 ul each, with a plasma dilution of 1:10. Concentrations of Exenatide were subsequently determined from standard curves of newly prepared Exenatide, following preliminary studies to ensure that all results fell within the linear range of the plasma standard curves. Notably, this Exenatide assay appears to lack cross-reactivity with glucagon, oxyntomodulin, GLP-1 or GLP-2.28. In brief, ELISA plates were coated with Exendin-4 (concentration: 2 ug/ml in coating buffer) at 4 °C overnight, and following blocking and washing steps, standards  and unknown samples with serial dilutions were added to the plate and incubated at room temperature for 1 h. After washing, biotinylated-Exenatide (concentration: 2 ug/ml) was added, and was followed by washing and SA-HRP detection. The titers of the anti-Exenatide antibody within the samples were then estimated by serial dilution of the plasma (to a maximum dilution of 1:225). We used results from the 1:25 dilution of all samples to determine whether a positive or negative development of anti-Exenatide antibodies had occurred across all 12 nonhuman primates evaluated in our study.As both PT320 and Bydureon are slow-release formulations of Exenatide and our study extended over a period of 12 weeks, we evaluated plasma anti-Exenatide antibody levels at both week 6 and week 12 using a previously developed homemade Sandwich ELISAIpeak) associated with the initial release burst of Exenatide, and the time to reach CIpeak (TIpeak). (ii) The lowest concentration of Exenatide in plasma (CItrough) achieved subsequent to the CIpeak, and the time of this decline (TItrough). (iii) The ultimate maximal Exenatide concentration (CMax), and time to reach it (TMax). (iv) The lag time between the achievement of CIpeak and CMax (TLag). (v) The area under the curve from 0 to the last time point (AUC0–last), as calculated by the linear trapezoidal and log-linear trapezoidal methods. The lower limit of quantitation (LLOQ) for Exenatide in our study was 3.1 pg/ml.The pharmacokinetic characteristics of PT320 and Bydureon were assessed by following Exenatide time-concentration profiles and pharmacokinetic parameters, which were analyzed with noncompartmental methods using WinNonlin . The baseline (zero time) sample was quantified for each animal as a level of ‘background signal’, and this value was then subtracted from time-dependent values in the same animal. The pharmacokinetic parameters evaluated included (i) the initial peak concentration (CCharacteristics of currently available US FDA approved GLP-1 receptor agonists


Indomethacin is a commonly used medication against preterm delivery. Several reports of fetal ductal constriction have been described after indomethacin use in the literature; however, there are no previously documented reports describing an association between Twin-Twin Transfusion syndrome and a constrictor effect of indomethacin on the ductus arteriosus. Two patients were referred to our department for Twin-Twin Transfusion syndrome and each underwent placental laser surgery. Constriction of the ductus arteriosus occurred as early as 20 and 24 weeks’ gestation following maternal use of indomethacin after laser surgery. Spontaneous amelioration was observed after discontinuation of the drug. The constrictor effect of indomethacin on the ductus arteriosus can be observed even after a single dose and as early as 20 weeks of gestation in complicated monochorionic twin pregnancies. We emphasize meticulous use of indomethacin in complicated monochorionic twin pregnancies because the constrictive effect seems to be independent of gestational age. Twin-Twin Transfusion syndrome (TTTS) is a complication of MC twin pregnancies and foetoscopic laser photocoagulation is one of the treatment options; however, laser surgery increases the risk of abortion/preterm delivery. Indomethacin is a commonly used medication against preterm delivery. Several reports of fetal ductal constriction have been described after indomethacin use in the literature. Here, we present two cases of antenatally diagnosed foetal ductal constriction in the donor co-twin after indomethacin use.Monochorionic (MC) twins are a subtype of monozygotic (MZ) pregnancies and are seen in 0.3% of all pregnancies. MC diamniotic pregnancies are the most common subtype of MZ pregnancies and account for 70-75% of all MZ pregnanciesth percentile)  (2). Theral shape . The trith centile for donor twin. Placental laser surgery was performed after preoperative counselling. Postoperatively, a single-dose 100 mg indomethacin suppository was inserted into the rectum to prevent uterine contractions. On the next day (postoperative day 1), constriction of the ductus arteriosus and tricuspid regurgitation were observed in the donor twin. There was a marked ductal narrowing  was less than 10arrowing . The peaarrowing . On postarrowing . On post. The diagnostic criteria of ductal constriction are as follows: a) presence of turbulent flow with a continuous pattern in the ductus arteriosus region (systolic and diastolic) in colour Doppler; b) systolic velocity ≥1.4 m/s; and c) diastolic velocity ≥0.3 m/s. To the best of our knowledge, this is the first report of prenatal diagnosis of constriction of ductus arteriosus in as early as 20 and 24 weeks’ gestation following maternal use of indomethacin in complicated MC twins after laser surgery. Prostaglandin inhibitors such as indomethacin are tocolytic agents, which act by decreasing prostaglandin activity in the uterine myometrium. However, decreased prostaglandin synthesis in the foetal circulation may have adverse effects on the ductus arteriosus because prostaglandins mainstay ductal patency. Fetal ductal constriction is a distinct entity caused by different drugs ; however, idiopathic/spontaneous constriction of foetal ductus arteriosus has also been described in the literature.Tricuspid regurgitation, right ventricular dilatation, oligohydramnios, ascites, hydrops, and increased nuchal translucency are common foetal findings in such cases. A case series reported 45 cases of fetal ductus arteriosus constriction and closure, including 8 idiopathic forms. The most common finding of ductal constriction was tricuspid regurgitation, present in 86.6% of patients. Although indomethacin was administered as a single dose at 20 weeks’ gestation and once a day for 2 days at 24 week’s gestation, our cases presented with constriction of the ductus arteriosus. As we know from previous reports, constriction of the ductus arteriosus is not dependent on serum indomethacin levels. Vermillion et al. analysed the effect of indomethacin tocolysis on fetal ductus arteriosus constriction of 72 fetuses and reported that the greatest incidence of ductal constriction occurred at 31 weeks’ gestation. The earliest gestational age for ductal constriction was reported at 24.7 weeks’ gestation. Therefore, it could be argued that the ductal effect of indomethacin is unpredictable for the foetus at any gestational age. Complete and irreversible closure of the ductus arteriosus can be observed even after a single dose of indomethacin, whereas ductus arteriosus can remain unaffected despite repeated doses. In our cases, constriction of the ductus arteriosus was seen only in the donor twin. In our opinion, this effect was observed only in the donor twins because of the relatively hypoxic conditions of donor foetuses. A selective constrictive effect of indomethacin on MC twins has been shown previously in some pathologically proven reports. Furthermore, indomethacin can decrease fetal urine output and lead to deterioration in amniotic fluid levels in the donor foetus. Thus, it seems reasonable to use tocolytics other than indomethacin in TTTS. Just a few cases of hydrops and constriction of ductus arteriosus following indomethacin use have been described in the literature and none presented as early as 24 weeks’ gestation. As in our case, hydrops resolved after discontinuation of indomethacin. Indomethacin use is associated with a risk of constriction of the ductus arteriosus at any gestational age; however, this condition and its effects can present in the absence of Doppler criteria. In case 1, the absence of Doppler criteria can be explained by the relatively early gestational age and severe tricuspid regurgitation. Tricuspid regurgitation allows the right ventricle to empty and may prevent turbulent flow in the ductus arteriosus. In conclusion, these are the first reported cases of foetal ductal constriction in complicated MC twin pregnancy presenting as early as 20 weeks’ of gestation. Therefore, we emphasize meticulous use of indomethacin in complicated MC twin pregnancies because the constrictive effect seems to be independent of gestational age. In the event of indomethacin use, Doppler indices of the ductus arteriosus should be closely monitored.Preterm delivery is a major cause of perinatal morbidity and mortality. Indomethacin is relatively old and the most commonly used prostaglandin synthetase inhibitor, it has been used in preterm delivery since the 1970s. Maternal use of indomethacin is the most common cause of foetal ductus arteriosus constriction; however, idiopathic constriction of the fetal ductus arteriosus has also been described in the literature


The down‐regulation of ABCA1 in sEH−/− macrophages was due to an increase in the turnover rate of ABCA1 protein but not in mRNA transcription. Inhibition of phosphatase activity, but not hydrolase activity, of sEH decreased ABCA1 expression and cholesterol efflux following oxLDL challenge, which resulted in increased cholesterol accumulation. Additionally, oxLDL increased the phosphatase activity, promoted the sEH‐ABCA1 complex formation and decreased the phosphorylated level of ABCA1 at threonine residues. Overexpression of phosphatase domain of sEH abrogated the oxLDL‐induced ABCA1 phosphorylation and further increased ABCA1 expression and cholesterol efflux, leading to the attenuation of oxLDL‐induced cholesterol accumulation. Our findings suggest that the phosphatase domain of sEH plays a crucial role in the cholesterol metabolism of macrophages.The contribution of soluble epoxide hydrolase (sEH) to atherosclerosis has been well defined. However, less is understood about the role of sEH and its underlying mechanism in the cholesterol metabolism of macrophages. The expression of sEH protein was increased in atherosclerotic aortas of apolipoprotein E‐deficient mice, primarily in macrophage foam cells. Oxidized low‐density lipoprotein (oxLDL) increased sEH expression in macrophages. Genetic deletion of sEH ( Our second aim was to characterize the role of sEH in oxLDL‐deregulated cholesterol metabolism and the underlying molecular mechanism in bone marrow‐derived macrophage (BMDM) foam cells by use of loss‐of‐function and gain‐of‐function strategies. Here, we report that the PT activity, but not the EH activity, of sEH plays a crucial role in regulating ABCA1‐dependent cholesterol efflux during the transformation of macrophage foam cells. Our findings provide a novel explanation for the anti‐atherogenic properties of sEH and suggest a molecular target for the treatment of atherosclerosis.Given the importance of sEH in the development of CADs, in this study we investigated the role and the mechanism of sEH in regulating the cholesterol metabolism of macrophage foam cells. We first investigated the expression and distribution of sEH in atherosclerotic lesions of apolipoprotein E null , and goat antibodies for SR‐A and ABCG1 were from Santa Cruz Biotechnology. Mouse antibodies for ABCA1, rabbit antibodies for SR‐BI and F4/80 were from Abcam. 12‐(3‐adamantan‐1‐yl‐ureido)‐dodecanoic acid (AUDA), NBD cholesterol, oleic acid (OA) and TBARS assay kits were from Cayman Chemical. Cholesterol and triglyceride assay kits were from Randox. Peroxidase‐conjugated antimouse IgG, anti‐rabbit IgG, anti‐goat IgG and anti‐rat IgG antibodies were from Jackson ImmunoResearch.2.2Apoe−/− mice and EPXH2−/− sEH‐deficient (sEH KO) mice were obtained from the Jackson Laboratory. Mice were housed in barrier facilities on a 12‐hour light/12‐hour dark cycle and fed with regular chow .All animal experiments were approved by the Animal Care and Utilization Committee of National Yang‐Ming University. C57BL/6 mice were purchased from the National Laboratory Animal Center, National Science Council, and 2.32O2 for 10 minutes. After blocking with BSA, slides were incubated with primary antibodies for 1 hour at 37°C and with corresponding secondary antibodies for an additional 1 hour. Antigenic sites were visualized by the addition of DAB. Slides were counterstained with haematoxylin.Formalin‐fixed, paraffin‐embedded tissue blocks were cut into 8 µM sections. Sections were deparaffinized, rehydrated and covered with 3% H2.42 inhalation and mononuclear cells from their femurs were harvested by Percoll (1.073 g/cm3) density‐gradient centrifugation. The cells were then seeded in MEMα supplemented with 50 ng/mL macrophage colony‐stimulating factor, 10% FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) for differentiation for 5 days at 37°C. The differentiated macrophages were then subjected to further experiments. HEK293 cells and Huh7 hepatoma cells were cultured in DMEM supplemented with 10% FBS and penicillin (100 U/mL)/streptomycin (100 µg/mL) at 37°C.Bone marrow‐derived macrophages were prepared as previously described.2.54 for 24 hours at 37°C, and Cu2+ was then removed by extensive dialysis. The extent of modification was determined by measuring thiobarbituric acid‐reactive substances (TBARs). OxLDL containing approximately 30‐60 nmol of TBARs, defined as malondialdehyde equivalents per mg of LDL protein, was used for experiments.The oxLDL was prepared as described previously.2.6g for 10 minutes. This cell lysate was added to 4‐nitrophenyl phosphate to 2 mM and incubated at 37°C for 1 hour. The yellow colour product was detected by OD405 nm to determine PT activity.Bone marrow‐derived macrophages with or without indicated treatments were collected in phosphate‐buffered saline (PBS), sonicated, and the supernatant was collected by centrifugation at 10 000 2.7Cells were fixed with 4% paraformaldehyde and then stained by 0.5% oil red O. Haematoxylin was used as a counterstain. The intracellular lipid content was evaluated by alcohol extraction after oil red O staining. The absorbance at 540 nm was measured using a microplate reader (BioTek Instruments).2.8Cellular cholesterol and triglyceride were extracted by hexane/isopropanol . After removing cellular debris, the supernatant was dried under nitrogen. The levels of cholesterol and triglyceride were measured using cholesterol and triglyceride assay kits (Randox).2.9g for 10 minutes, washed 3 times with cold PBS and then eluted in sodium dodecyl sulphate (SDS) lysis buffer . Eluted protein samples were separated by 8% or 10% SDS‐PAGE. After transfer to membranes, the samples were incubated with primary antibodies, washed and then incubated with secondary antibodies conjugated with horseradish peroxidase. Bands were revealed using an enzyme‐linked chemiluminescence detection kit (PerkinElmer), and signals were quantified using Imagequant 5.2 software .The methods for immunoprecipitation and Western blot analysis as previously described.2.10Macrophages were equilibrated with NBD cholesterol (1 µg/mL) for 12 hours. These cells were washed with PBS and incubated with oxLDL (50 µg/mL) in RPMI 1640 medium for another 12 hours in the presence of apoAI (10 µg/mL) or HDL (50 µg/mL). The fluorescence‐labelled cholesterol released from the cells into the medium was analysed using a multilabel counter (PerkinElmer) with 485 nm excitation and 535 nm emission.2.11Taq DNA polymerase (Geneaid) as follows: 2 minutes at 94°C, 15 seconds at 94°C, 30 seconds at 58°C and 1 minutes at 72°C for 35 cycles. The primers for full‐length sEH were 5′‐TTACGCGTGCGCTGCGTGTAGCCG‐3′  and 5′‐GGTCTAGACTAAATCTTGGAGGTCACTG‐3′ . The primers for the N‐terminal PT domain were 5′‐TTACGCGTGCGCTGCGTGTAGCCG‐3′ (forward with MluI site) and 5′‐GGTCTAGACTACCCTGTGACCTTCTCCA‐3′ (reverse with XbaI site). The primers for the C‐terminal EH domain were 5′‐TTACGCGTGTCAGCCATGGATATGTGAC‐3′ and 5′‐GGTCTAGACTAAATCTTGGAGGTCACTG‐3′. PCR products were cloned into the pGEM vector according to the manufacturer's instructions (Promega). Clones were confirmed by sequencing. These three plasmids were digested with MluI and XbaI, and inserts were ligated into the pCMV5 N‐Flag vector. A replication‐defective recombinant adenoviral vector containing a human phosphoglycerate kinase (hPGK) promoter driving the human EH domain (Adv‐EH) or PT domain (Adv‐PT) of sEH, as well as hPGK alone to serve as a control (Adv‐null), was constructed. Recombinant adenovirus was generated by homologous recombination, amplified in HEK293 cells, purified by CsCl ultracentrifugation, and stored in 10 mm Tris‐HCl (pH 7.4), 1 mm MgCl2 and 10% (vol/vol) glycerol in liquid nitrogen until used for experiments. The titres of adenovirus were determined by plaque assay in HEK293 cells. Macrophages were infected with 50 MOI of adenovirus for 24 hours and then subjected to experiments.C‐terminal hydrolase and N‐terminal phosphatase domains of sEH were isolated by polymerase chain reaction (PCR) with different primer sets from a human sEH cDNA clone. PCR was performed with Hi Fi 2.12U test was used to compare 2 independent groups. The Kruskal‐Wallis followed by Bonferroni post hoc tests was used to account for multiple comparisons. SPSS v 20.0 (SPSS Inc) was used for analysis. Differences were considered statistically significant at P < 0.05.Data are presented as mean ± SEM from 5 mice or 5 independent cell experiments. Data from animal studies were evaluated by parametric tests. One‐way ANOVA followed by the LSD test was used for multiple comparisons. Data from cell studies were evaluated by non‐parametric tests. The Mann‐Whitney 33.1Apoe−/− mice. Western blot analyses demonstrated that the expression of sEH was increased in the aortas of Apoe−/− mice relative to those of control WT mice  or ebselen (an inhibitor of sEH PT activity). As shown in Figure 3.6Previous studies have reported the phosphorylation status of ABCA1 protein is crucial for its stability.To address whether hydrolase activity or PT activity of sEH is involved in the changes of ABCA1 phosphorylation caused by oxLDL challenge, we amplified the EH activity or PT activity of sEH by overexpressing the PT or EH domains of sEH using an adenovirus system. As shown in Figure 4In this study, we identified a novel role of PT activity of sEH in cholesterol homeostasis during the transformation of macrophage foam cells. We first determined that sEH was significantly increased in atherosclerotic aortas and in particular, in intralesional macrophage foam cells. In addition, we found that oxLDL, the most critical atherogenic factor in early atherosclerosis, increased the expression of sEH. These results suggested a role for sEH in the lipid metabolism of macrophage foam cells. Genetic manipulations and pharmacological inhibition revealed that oxLDL‐mediated ABCA1 expression is regulated by the PT activity of sEH. We demonstrated that genetic deletion of sEH or pharmacological inhibition of PT activity, but not inhibition of EH activity, augmented oxLDL‐induced lipid accumulation in macrophages. Moreover, exposure of macrophages to oxLDL increased the physical interaction of sEH with ABCA1 and decreased ABCA1 phosphorylation. More importantly, inhibition of sEH PT activity by ebselen or genetic deletion of sEH prevented the increase in ABCA1 protein expression and cholesterol efflux following oxLDL treatment. By contrast, overexpression of the PT domain of sEH decreased ABCA1 phosphorylation by oxLDL and consequently increased the ABCA1 protein level and cholesterol efflux, leading to the attenuation of lipid accumulation in macrophage foam cells. Together, these results suggest that sEH PT activity has a unique function in regulating cholesterol clearance in macrophage foam cells.Apoe−/− mice increases cholesterol efflux from macrophages and slows down the progression of atherosclerosis.Accumulation of macrophage‐derived foam cells in the intima of the aorta is a critical event for the initiation and progression of atherosclerosis.Under physiological conditions, sEH is an enzyme with the C‐terminal EH activity and N‐terminal PT activity.Our results showed the inhibition of EH activity by AUDA had no effect on basal level of cholesterol efflux or oxLDL‐induced cholesterol efflux and lipid accumulation in macrophages. These data are inconsistent with those of Shen et alEPXH2−/− sEH‐deficient mice, it is difficult to determine which domain contributes to the alterations in physiological functions under in vivo conditions. Therefore, we used an in vitro model to isolate the role of PT activity. In our previous studies, we found PT activity of sEH plays a crucial role in vascular endothelial growth factor (VEGF)– and simvastatin‐induced activation of endothelial nitric oxide synthase (eNOS).In contrast to the EH activity, less is known about the biological roles of the PT activity of sEH. Whether PT activity participates in the regulation of cholesterol metabolism and its underlying mechanisms is not fully understood. The PT activity of sEH is assumed to prefer phosphates on lipophilic compounds as substrates.With respect to the function of sEH in cholesterol metabolism, whether EH or PT activity is involved is still not clear. Based on work by EnayetAllah et alThe critical role of ABCA1, the major transporter responsible for the apoAI‐dependent cholesterol efflux, in regulating cholesterol homeostasis in macrophages has been well‐characterized.Under physiological condition, the lipid homeostasis of whole body is tightly controlled by the cross‐talk between liver, adipose tissues and peripheral tissues including vascular system. Nevertheless, our study contains several limitations because we only examined the role of PT activity of sEH in cholesterol metabolism of macrophage foam cells. From the point in search of drug targets, the role of PT activity of sEH in lipid metabolism should be investigated in more cell types such as hepatocytes and adipocytes. In addition, the role of PT activity of sEH in overall lipid metabolic balance under physiological or pathological conditions should be investigated in vivo. To this end, further investigations describing the implications of PT activity of sEH in lipid homeostasis and metabolic diseases are warranted.In conclusion, we provide new evidence for PT activity of sEH in the regulation of cholesterol metabolism in macrophage foam cells. The PT activity of sEH reduces oxLDL‐induced ABCA1 phosphorylation and stabilizes ABCA1, which resulted in increased cholesterol efflux and decreased lipid accumulation in macrophage foam cells. Activation of the PT of sEH may be a pharmacological target for atherosclerosis and related cardiovascular diseases.The authors declare that there are no conflicts of interest.CC Lien, CH Chen, YM Lee, BC Guo, LC Cheng and CC Pan performed the experiments and analysed the data; SK Shyue and TS Lee designed the experiments and wrote the paper. Click here for additional data file.


Electrospun xanthan polysaccharide nanofibers (X) were developed as an encapsulation and delivery system of the poorly absorbed polyphenol compounds, gallic acid (GA) and (−)-epigallocatechin gallate (EGCG). Scanning electron microscopy was used to characterize the electrospun nanofibers, and controlled release studies were performed at pH 6.5 and 7.4 in saline buffer, suggesting that the release of polyphenols from xanthan nanofibers follows a non-Fickian mechanism. Furthermore, the X-GA and X-EGCG nanofibers were incubated with Caco-2 cells, and the cell viability, transepithelial transport, and permeability properties across cell monolayers were investigated. Increases of GA and EGCG permeabilities were observed when the polyphenols were loaded into xanthan nanofibers, compared to the free compounds. The observed in vitro permeability enhancement of GA and EGCG was induced by the presence of the polysaccharide nanofibers, which successfully inhibited efflux transporters, as well as by opening tight junctions. Polyphenols are the most abundant antioxidants in our diet and they are receiving increasing interest due to the established association between the intake of a polyphenol-rich diet and the prevention of various diseases ,2. BecauPhyllanthi tannin fraction at a dose of 6 g/kg in rats, the maximum concentration of absorbed GA was less than 10.47 µg/mL  (ATCCGermany) . w/v under vigorous stirring overnight at room temperature. Subsequently, GA and EGCG were added to the polysaccharide solution and further stirred for 30 min. The electrospinning setup consisted of a high voltage generator  to provide a voltage of 20 kV, and a syringe pump  to feed the xanthan solution at a flow rate of 0.01 mL/min using a 21 G needle gauge. Xanthan fibers were collected on a steel plate covered with an aluminum foil perpendicularly placed at 8 cm from the end of the needle. The electrospinning process was carried out at ambient conditions (20°C and around 20% relative humidity).Xanthan was dissolved in formic acid at a final concentration of 2.5% The morphology of electrospun X, X-GA, and X-EGCG nanofibers was studied using a Phenom Pro scanning electron microscope (SEM) . For SEM analysis, a small piece of nanofibers web was attached on SEM specimen stubs by a double-sided adhesive tape. The average fiber diameter of nanofibers was calculated using image J analysis software  measured at 100 different points for each image.−1. The infrared peaks were identified with a Spectrum™ 10 software using a 1% transmittance (T) peak threshold.Fourier transform infrared (FTIR) spectroscopy analysis of X, X-GA, X-EGCG nanofibers, GA, and EGCG was analyzed using a Perkin Elmer Spectrum 100 spectrometer  based on a universal attenuated total reflectance (ATR) sensor. Four scans for each sample were accumulated at 20 °C at a resolution of 1 cmThe amount of GA and EGCG loaded into xanthan nanofibers was evaluated by immersing the nanofibers in equal volumes of complete growth medium (DMEM-FBS) or HBSS at pH 6.5 or pH 7.4. Briefly, 1.0 mg of X-GA and X-EGCG fibers were immersed in 2 mL pre-warmed medium in a 48-well plate, and the release of molecules from nanofibers was conducted at 37 °C for 8 h. The withdrawn aliquots were analyzed by RP-HPLC with detection of GA and EGCG at 255 nm and 270 nm, respectively see also . The cum5 cells/mL in T-75 cm2 flasks and incubated at 37 °C in a humidified atmosphere of 5% CO2. The complete cell medium, here indicated as DMEM-FBS, consisted of high glucose DMEM containing 10% heat-inactivated FBS, 2 mM l-glutamine, 1% nonessential amino acids, 100 U/mL penicillin, and 100 µg/mL streptomycin. The medium was renewed every second day until cells reached approximately 90% confluence. Cells were passaged at a subcultivation ratio of 1:4 by treatment with 0.25% trypsin—0.53 mM EDTA solution for 10 min at 37 °C. After trypsinization, the cells were suspended in complete growth medium and centrifuged for 5 min at 1000 rpm. After supernatant removal, the pellet was suspended in the growth medium and cell concentration was determined with an ORFLO Moxi Z Mini Automated Cell Counter using Type S cassette . All Caco-2 cells were used between passages 9–15.Caco-2 cells were routinely seeded at a concentration of 1.0 × 10app) after GA and EGCG release from nanofibers and as free compounds. Before testing nanofiber mats with Caco-2 cells, the collected fibers were kept under an air stream for 3 days allowing complete formic acid evaporation. Besides GA and EGCG, the transepithelial transport of fluorescein (FLUO) and Lucifer yellow (LY) across Caco-2 cell monolayers were also investigated as markers for intestinal epithelial permeability and integrity.Xanthan (X), gallic acid-loaded xanthan (X-GA), and (−)-epigallocatechin gallate-loaded xanthan (X-EGCG) nanofibers were produced by electrospinning a solution of the mixed compounds dissolved in formic acid. These nanofibers were tested with Caco-2 cell monolayers to evaluate their toxicity and apparent permeability coefficient  was recorded at 490 nm through a well plate reader .The in vitro Caco-2 cell viability after treatment with free GA, free EGCG, xanthan nanofibers (X), GA-loaded xanthan nanofibers (X-GA), and EGCG-loaded xanthan nanofibers was evaluated by using the MTS [3--5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt] colorimetric bioassay. Different concentrations of free GA and EGCG ranging from 1 µM to 1 mM were prepared in PBS and sterile-filtered with a 0.22 µm pore size. Furthermore, increasing amounts of dried X, X-GA, and X-EGCG nanofibers were peeled off from the aluminum foils and incubated with cells. In a 48-well plate, 1.5 × 105 cells/insert were seeded onto pre-wetted 12-mm polycarbonate cell culture inserts with an area of 1.12 cm2 and a pore size of 0.4 µm. The apical and basolateral compartments were filled with 0.5 mL and 1.5 mL complete medium, respectively. The Caco-2 cells were incubated onto the filters overnight at 37 °C in a humidified atmosphere of 5% CO2. The day after, the growth medium was replaced in both compartments and the plates were incubated for 21 days at 37 °C in a humidified atmosphere of 5% CO2, renewing the complete growth medium every second day. For the AB transport experiments, donor solutions of FLUO, LY, GA, and EGCG at a concentration of 11 mM, 9.57 mM, 1.1 mM, and 1.1 mM, respectively, were prepared in sterile-filter HBSS at pH 6.5 buffered with 10 mM MES. Again, donor solutions of FLUO, LY, GA, and EGCG at a concentration of 10.3 mM, 9 mM, 1.03 mM, and 1.03 mM, respectively, were prepared in sterile-filter HBSS at pH 7.4 buffered with 25 mM HEPES to evaluate their BA transport. A volume of 50 µL of each stock solution was added to the donor chamber . The transport of GA and EGCG released from nanofibers and as free compounds in the presence of empty X nanofibers was also investigated. For the AB transport, 0.2 mg X-GA, 1.0 mg X-EGCG, 0.2 mg, and 1.0 mg X were used, and accordingly, 0.6 mg X-GA, 3.0 mg X-EGCG, 0.6 mg, and 3.0 mg X were incubated with cell monolayers to evaluate their BA transport. Prior to incubation of the nanofibers, the mats were peeled off from the aluminum foil and kept under an air stream for 3 days. After 21 days of cell growth, the complete DMEM medium was removed from the cell monolayers and replaced with HBSS at pH 6.5 and pH 7.4 at the apical and basolateral compartments, respectively. For the AB transport studies, 1.5 mL HBSS was used in the basolateral side and 0.55 mL of each donor solution and/or nanofibers were added to the apical side. Immediately, 200 µL aliquots were withdrawn from each donor compartment (time = 0). Aliquots from the acceptor side were then withdrawn at different time intervals, and the volume was replaced with fresh HBSS at pH 7.4 maintaining the well plates at 37 °C in a humidified atmosphere of 5% CO2. A final aliquot from the donor chamber was taken as the last time point. BA transport studies were conducted using the same procedure and incubating 0.5 mL HBSS at pH 6.5 in the apical side and 1.55 mL of donor solution and/or nanofibers in the basolateral chamber. During the transport experiments, all cell media were pre-warmed at 37 °C. Each transport experiment was performed for a time interval of 8 h in triplicate (n = 3). After 8 h of transport studies and TEER measurements, both apical and basolateral chambers were washed twice with PBS and cell monolayers were detached from the insert membrane with 0.25% trypsin-0.53 mM EDTA solution for 10 min at 37 °C. The collected Caco-2 cell lysates were centrifuged for 5 min at 1000 rpm and supernatants were discarded. Furthermore, the semipermeable membranes were carefully removed from the insert using a scalpel and collected into Eppendorf tubes in 500 µL HBSS at pH 6.5 . Cell pellets were re-suspended in 500 µL HBSS at pH 6.5 and both cells and membranes were sonicated for 3 h using an ultrasonic bath . The collected samples were then centrifuged for 15 min at 10,000 rpm and supernatants were analyzed by HPLC . The same procedure was used to quantify the compound amounts adsorbed (X + GA and X + EGCG) or remaining encapsulated (X-GA and X-EGCG) in the nanofibers at the end of the transport experiments. The tested nanofibers were removed from the donor chamber and suspended in 500 µL of HBSS (pH 6.5 for AB transport and pH 7.4 for BA transport). After sonication and centrifugation, the molecules in the supernatants were quantified by HPLC.The transepithelial transport of free fluorescein (FLUO), free lucifer yellow (LY), free gallic acid (GA), free (−)-epigallocatechin gallate (EGCG), free gallic acid in the presence of empty xanthan nanofibers (X + GA), free (−)-epigallocatechin gallate in the presence of empty xanthan nanofibers (X + EGCG), gallic acid-loaded xanthan nanofibers (X-GA), and (−)-epigallocatechin gallate-loaded xanthan nanofibers (X-EGCG) across Caco-2 cell monolayers were investigated according to the protocol reported by Hubatsch et al. . The traBLANK) were recorded and subtracted from the resistance values obtained from the measurement of each cellular monolayer onto the semipermeable membrane (RTOTAL). The specific cell resistance values (RTISSUE) were calculated by:The transepithelial electrical resistance (TEER) was measured at 20 °C before and after permeability experiments with an epithelial volt-ohmmeter equipped with STX2 “chopstick” electrodes . Before measuring the resistance values of each well, the cell monolayers and the basolateral chamber were washed twice with pre-warmed HBSS at pH 6.5 and HBSS at pH 7.4, respectively. The resistance values of the semipermeable membrane without cells , recording their absorbance at 490 nm and 430 nm, respectively. The amount of each compound transported across the cell monolayers within a time interval of 8 h was calculated for both apical-to-basolateral (AB) and basolateral-to-apical (BA) directions. FLUO, LY, GA, and EGCG that remained entrapped within the cell monolayers, insert membranes and nanofibers were likewise quantified at the end of the permeability studies.Donor solutions of FLUO, LY, GA, and EGCG were prepared and sterile-filtered in HBSS at pH 6.5 and pH 7.4 to perform transepithelial studies. Standard curves of GA and EGCG dissolved in HBSS at pH 6.5 and pH 7.4 were obtained by HPLC analysis. 200 µL samples withdrawn from the donor and acceptor compartments during transport experiments across cell monolayers were quantitatively analyzed using RP-HPLC . A C18 column (3.0 × 100 mm) and 0.5 mL/min flow rate were used. GA and EGCG were quantified with detection at 255 nm and 270 nm, respectively. FLUO and LY aliquots were instead analyzed by UV-vis spectrometry , donor and acceptor concentrations at time = 8 h , compound concentrations remaining inside the cell monolayer at time = 8 h , within membrane filters at time = 8 h , and adsorbed or remaining encapsulated in nanofibers at time = 8 h  were experimentally measured. The mass balance of each compound was calculated as follows:After transport experiments in both AB and BA directions, the amount of each compound collected at the apical and basolateral chambers was quantified. Donor concentrations at time = 0  and the secretory apparent permeability coefficient  were calculated by:dC/dt (µM/s) is the rate of change in concentration on the acceptor chamber over time; V (cm3) is the volume of the solution in the acceptor compartment; A (cm2) is the area of the semipermeable membrane; and C0 (µM) is the initial concentration in the donor chamber. The results of this study are expressed as mean ± SD of three independent experiments. Permeability directional ratio (PDR) is a measure of the compound polarization in Caco-2 cell monolayers, and was calculated by:The absorptive apparent permeability coefficient , HBSS at pH 6.5 and HBSS at pH 7.4 . The totC is the amount of the compound released within the time, t; k is the rate constant; and n the release exponent. The constant value of k is usually related to the characteristics of the delivery system and drug; while n is the diffusion exponent, which characterizes the transport mechanism of the compound, and it depends on the type of transport, geometry, and polydispersity. The n values of the kinetic model in pH 6.5 and 7.4 media for the release of GA were 0.85 and 0.83, respectively. In the case of EGCG release, the n values in pH 6.5 and 7.4 media were 0.84 and 0.77, respectively. These results confirm that the release of the studied phenolic compounds is governed by the non-Fickian mechanism.The mechanism of GA and EGCG release from X nanofibers in pH 6.5 and 7.4 media were fitted by a Korsmeyer-Peppas kinetic model  to be used for transepithelial transport studies. As shown in 2. The TEER values of monolayers before and after transport of FLUO and LY were found in the range of 300–500 Ω cm2 , in a free form in the presence of empty xanthan nanofibers (X + GA and X + EGCG), and in the nanofiber forms (X-GA and X-EGCG). ectively C. The saectively E. An incThe fate of GA and EGCG during 8 h transepithelial transport in both AB and BA directions, was monitored by quantifying their concentration in the donor and acceptor compartments, in the cell lysate, insert membrane (filter), and within xanthan nanofibers (adsorbed or unreleased). In this study, human differentiated epithelial Caco-2 cells were chosen as an established in vitro cell model for the prediction of bioactive compounds’ absorption and transport mechanism . The CacThe incubation of nanofibers with Caco-2 cells (24 h) revealed a proliferative effect in cell viability for amounts lower or equal to 0.5 mg X-GA and 2.0 mg X-EGCG; a drastic cell mortality was observed for doses above this range. In addition, the treatment of Caco-2 cells with increasing amounts of empty xanthan nanofibers resulted in a dose-dependent reduction of cell viability, close to 60% viability for 10.0 mg X incubated. However, the observed reduction in cell viability was expected to be less pronounced for transepithelial transport studies, since the cell monolayers were exposed to X, X-GA, and X-EGCG for 8 h rather than 24 h. The transepithelial transport of GA and EGCG in the acceptor compartment was successfully enhanced by the presence of xanthan, both as an empty nanostructure and as a nanocarrier, and the permeability coefficients were higher than those calculated for free compounds. In addition, the PDR values of free GA and free EGCG were both higher than 1.5 , suggesting that their transport is modulated by an active transport pathway, and more specifically by efflux. Several studies have described the mechanism and the efflux transporters involved in the unidirectional transport of GA and EGCG across the epithelial barrier ,19,50,51The results presented in this study are congruent with our previous findings on the permeation across Caco-2 cells of a model protein (insulin) encapsulated within electrospun fish protein fibers . Direct Encapsulation and release of two poorly absorbed polyphenol compounds, GA and EGCG, using electrospun xanthan nanofibers were investigated. It was found that X, X-GA, and X-EGCG nanofibers remained stable in aqueous HBSS medium at different pH (6.5 and pH 7.4). The total amount of GA and EGCG released from xanthan nanofibers was 69.01% and 70.53% in HBSS at pH 6.5, and 58.47% and 83.44% in HBSS at pH 7.4, respectively. Moreover, the nanofibers were incubated with Caco-2 cells and the cell viability, transepithelial transport, and GA and EGCG permeability properties across cell monolayers were investigated. At least a 2-fold increase of GA and EGCG permeability was observed in the presence of X-GA and X-EGCG nanofibers, in comparison with the free-phenolic compounds. Indeed, the polysaccharide nanofibers enhanced the GA and EGCG permeability by opening the tight junctions of Caco-2 monolayers, as well as inhibiting the efflux transporters. These findings are relevant for promoting the delivery not only of polyphenols, but also of other poorly absorbable bioactives and drugs.