It is also recognized as an appealing alternative to other natural gas purification technologies such as adsorption and cryogenic processes due to its low cost, low energy requirement, easy membrane fabrication process and less requirement for supervision. During the past few decades, membrane-based gas separation technology employing hollow fibers (HF) has emerged as a leading technology and underwent rapid growth. Moreover, hollow fiber (HF) membranes have many advantages including high specific surface area, fewer requirements for maintenance and pre-treatment. However, applications of hollow fiber membranes are sometimes restricted by problems related to their low tensile strength as they are likely to get damaged in high-pressure applications. In this context, braid reinforced hollow fiber membranes offer a solution to this problem and can enhance the mechanical strength and lifespan of hollow fiber membranes. The present review includes a discussion about different materials used to fabricate gas separation membranes such as inorganic, organic and mixed matrix membranes (MMM). This review also includes a discussion about braid reinforced hollow fiber (BRHF) membranes and their ability to be used in natural gas purification as they can tackle high feed pressure and aggressive feeds without getting damaged or broken. A BRHF membrane possesses high tensile strength as compared to a self-supported membrane and if there is good interfacial bonding between the braid and the separation layer, high tensile strength, i.e., upto 170Mpa can be achieved, and due to these factors, it is expected that BRHF membranes could give promising results when used for the purification of natural gas.Natural gas is an important and fast-growing energy resource in the world and its purification is important in order to reduce environmental hazards and to meet the required quality standards set down by notable pipeline transmission, as well as distribution companies. Therefore, membrane technology has received great attention as it is considered an attractive option for the purification of natural gas in order to remove impurities such as carbon dioxide (CO It also contains small amounts of natural gas liquids which are called hydrocarbon gas liquids, as well as non-hydrocarbon gases such as water vapor and carbon dioxide (CO2). Natural gas is a widely used energy source that is used for heating, cooking, and generation of electricity, as well as fuel for vehicles. The purification of natural gas is important due to environmental hazards. Natural gas conditioning involves the elimination of acid gases such as CO2 and hydrogen sulphide (H2S), besides water vapor. The requirement for environment-friendly and energy-efficient gas purification techniques has encouraged extensive research into membrane-based gas purification technology. This technology is widely used in the separation of CO2 from natural gas. The removal of CO2 among other impurities is important because of its corrosive nature and its uncontrolled emission into the atmosphere has become a serious concern as it is hazardous for human health and also leads to climate change and flooding.Natural gas is formed deep beneath the earth’s surface and is a source of fossil energy. It consists of various compounds . Methane2. These technologies include adsorption, absorption, and membrane separation with their own advantages and disadvantages. Among available separation methods, membrane separation technology has come out to be a feasible option over other technologies because of advantages such as economy [Many different technologies were available on an industrial scale for natural gas purification to ensure the removal of CO economy ,4, proce economy . It was  economy , metal-o economy , carbon  economy , mesopor economy  were int economy . The inc economy . They exDuring the past few decades, membrane separation by employing hollow fiber (HF) membrane has become one of the most evolving technologies and experienced rapid growth. Hollow fiber membranes have managed to gain commercial interest with numerous applications at the forefront of experimental research in order to carry out purification of drinking water, bioseparations, and treatment of wastewater besides liquid phase and gaseous separations. Hollow fiber membranes are also playing a major role in gas separation applications due to them having high selectivity and separation areas. They are considered attractive for industrial use as they are self-supporting and can easily be assembled into modules ,13. HollThis review highlights the ways that could enhance the separation performance of membranes in natural gas purification. Mixed matrix membranes that comprise inorganic filler dispersed in a polymer matrix and combine the advantages of both polymeric and inorganic membranes offer an interesting approach to improving the separation performance. This review also highlights the fabrication of braid-supported HF membranes along with their applications. Moreover, it discusses the significance of polymer, braid and spinneret types employed for the fabrication of BRHF membranes and their effects on morphology as well as the performance of fabricated BRHF membranes.4) with other light gases such as butane (C4H10), propane (C3H8), ethane (C2H6) and corrosive gases such as hydrogen sulphide (H2S) and carbon dioxide (CO2) [Natural raw gas consists of methane . In this process, a blend of piperazine (PZ) and MDEA also known as activated MDEA (AMDEA) is mostly used [2.A reversible reaction among solvents and absorbed substances are involved in chemical absorption. Methyl diethanolamine (MDEA), monoethanolamine (MEA) and diethanolamine (DEA) are some widely recognized amines utilized as solvents for eliminating acidic gases , and methanol (CH3OH) are some different organic solvents used for the absorption of CO2. Genosorb® and Selexol® are the brand names of PEG liquids employed in organic physical scrubbing [2 in PEG is five times higher than in water [2 in the solvent. An organic physical scrubber is shown in This process is quite identical to water scrubbing, but instead of water, an organic solvent is used in this process. Polyethylene glycol ethers (PEG), crubbing . In the in water , which rin water . As comp2 and dissolved CH4 is discharged and recirculated towards the inlet of raw gas. For organic solvent regeneration, before entering the column of desorption it is heated further to about 40 °C. The solvent is injected into the column’s top and pressure is lowered to 1 bar. The solvent that is regenerated is then introduced to the top of the absorption column. The heat that is required in this process is waste heat and can be produced by regenerative thermal oxidation (RTO) unit and compressor which oxidizes CH4 in off-gas. Despite the fact that in terms of CO2 removal, this process is more efficient than water scrubbing but for the regeneration of solvent more energy is required. Moreover, the expense is higher for organic solvents than that for water [Before getting injected into the bottom of the absorption column the raw gas is cooled and compressed to 6–8 bars. To generate counter-current liquid and gas flow, the organic solvent is introduced from the column’s top. In order to maintain a low temperature of 20 °C in the absorption column, the organic solvent is also cooled before getting injected into the column. The organic solvent leaving the lower part of the absorption column is heat exchanged with the organic solvent which will be injected into the column’s top. The organic solvent is then injected into the flash column, where under reduced pressure some amount of COor water .2S and CO2 liquefies under different conditions of pressure and temperature. It works under the conditions of high pressure (80 bar) and low temperature (−170 °C). The boiling point of CO2 is −78.2 °C which is much higher than the boiling point of CH4, and liquefying CH4 allows CO2 to separate from CH4 [2S, siloxanes and other components that are not necessary are removed. The gas in the second step is compressed to 1000 kPa and cooled to −25 °C subsequently. The gas is further cooled until −55 °C in the third stage of this process and CO2 which is in the liquefied form is eliminated from the mixture of gas. The remaining stream of gas is then cooled further until −85 °C is achieved and CO2 reaches solid form which is then finally removed in the final and last stage. The refined gas is then depressurized and can be utilized in different applications. This separation system is viewed as an under-developed technique but some commercial plants are operational [2S and H2O is recommended to avoid equipment plugging caused because of water freezing in raw gas [The principle on which this separation is based is that different gas such as Hfrom CH4 ,40. A sefrom CH4 . For therational . If the rational . Pre-sep raw gas . The ope raw gas .2 at a faster rate than CH4 because of controlled diffusion rates or equilibrium type, adsorbing a huge load of CO2 than CH4. Commonly used materials include activated carbons, titanosilicates, carbon molecular sieves (CMS), natural and synthetic zeolites as well as silica gels. H2S must be removed from raw gas before introducing it to adsorption columns as it will bring irreversible damage to the adsorbents [2 is adsorbed while CH4 that is unaffected by adsorbent passes through the column. The inlet is closed when the column bed is full of CO2, and the blowdown phase begins. In order to desorb CO2 from the adsorbent, the pressure is reduced, and CO2-rich gas is expelled from the column. Along with the desorbed CO2, some methane is also lost. Finally, the purge phase begins at the lowest column pressure. In order to empty the column from the CO2 that is desorbed from the bed of the column, upgraded gas is blown through it. With an upgraded or raw gas, the column is regenerated and then repressurized [Pressure swing adsorption is a dry technique used to separate gases through contact between the adsorbent and the gas molecules. The adsorbents that are used have high specific areas to maximize the contact between gas and adsorbent and are porous solids. The adsorbents used are usually of kinetic type, adsorbing COsorbents . The pressurized .A PSA system usually consists of two to four columns as shown in Before the 1800s when synthetic nitrocellulose membrane was not prepared, biological membranes were made up of pig’s bladder, cattle, plant (onion skin) and fish . In 18312 such as pressure swing adsorption and cryogenic separation also suffer from problems related to energy consumption. Hence, the rapid and successful development of membrane-based gas separation technology has provided us with a convenient, energy-saving and economical separation process [Because of the extensive use of natural gas, the purification of natural gas has become a broad and wide-reaching gas separation process. Although the traditional amine absorption process undoubtedly can remove a high percentage of impurities, it still suffers from different drawbacks such as higher capital costs, higher energy consumption, complex operation and corrosion of equipment, etc. ,62. Like process .2 can permeate through the membrane and at the same time CH4 gets retained at the feed side as retentate as shown in 2 content at the inlet is high. These factors are quite favorable for standard natural gas purification units. The membrane-based gas separation technology is mostly used commercially because of its advantages such as better selectivity, low energy consumption as well as easily engineered modules. With this technology, high CO2 purity can be achieved along with efficient CH4 recovery up to 96%. This gas separation technology is based on gas dissolution and diffusion into polymeric membranes. Gas transportation across the film occurs by applying differential pressure on opposite sides of the polymer film. The rate of gas permeation is mostly controlled by the diffusion coefficient and solubility coefficient of the membrane system. Three types of membranes that are normally used for the gas separation process include polymeric, inorganic and mixed matrix membranes.In the case of purification of natural gas, CONormally in the field of membrane-based gas separations, attention is focused particularly on the permeability (productivity), as well as the selectivity (efficiency) of the membrane for a specified gas separation process . Hence, 4 purity was achieved by testing polyvinyl amine/polyvinyl alcohol blend membrane [2 and H2S [2 to get easily dissolved in the membrane matrix. PDMS (polydimethylsiloxane) due to its higher gas permeability in comparison with other synthetic polymers was viewed as a remarkable candidate [4 and CO2 in PDMS is relatively greater than others because of the existence of many configurations as well as the composition of the side chain. The obvious disadvantage of this kind of material is the low separation factor as well as low mechanical strength [Most of the membranes that are used commercially are polymeric and are made of organic materials such as cellulose acetate (CA), polysulfone (PSF), polydimethylsiloxane (PDMS), polycarbonate (PC) and polyimide (PI) ,67. Thesmembrane . For the and H2S . Cellulo and H2S . These m and H2S  because andidate . Permeabstrength .2/N2) selectivity is outlined as a function of the permeability of more permeable gas (CO2) as shown in 2 from a flue gas power plant would essentially require a polymer having a permeability of 1000 barrer at least, and selectivity of (CO2/N2) over 30 [In the past, several polymers have been synthesized and tested but unfortunately, only a few of them hit the market. Most of the commercial membranes are polymer-based and have low permeability and high selectivity. Low permeability can be related to the productivity of membrane-based gas separation process, hence, lower permeability makes them unsuitable for the treatment of large amounts of gas, for instance, treatment of flue gases. Though, the fabrication of membranes possessing higher permeability as well as selectivity is a challenging task because of the trade-off problem between permeability and selectivity. The trade-off was first proposed by Robeson , also it over 30 . At this over 30 ,76. In s2S and CO2 from natural gas, state-of-the-art polymeric membranes are competitive economically as compared to conventional technologies in operating and capital costs [In the separation of Hal costs . Table 2Inorganic membranes offer more thermal stability, are resistant to the chemicals and also offer better mechanical strength, so they are considered more advantageous than conventional polymeric membranes. They are normally made using zeolites, carbon molecular sieves (CMS), metal-organic frameworks and ceramics . As compInorganic membranes have many advantages such as solvent resistance at high-pressure conditions as well as stability at high-temperature conditions . Inorgan2S can negatively affect the performance of the membrane, so its pre-removal is necessary. In 2 as well as H2S from raw gas and can also trace the impurities using different membranes. As compared to the single-stage process, the multi-stage process has fewer operating and investment costs as well as gives high purity of CH4 [4 can be improved from 80 to 99.5% [In general, Hy of CH4 . Xiao ety of CH4  identifito 99.5% .The selection of the production process of the membrane depends on the desired membrane structure and the choice of polymer. Different methods are used to fabricate the membranes, these methods include; interfacial polymerization, phase inversion, track etching, controlled stretching, melt extrusion and electro-spinning . CurrentThis technique allows the synthesis of HF and flat sheet membranes. Phase inversion is generally a de mixing process in which polymer solution is transformed into the solid phase under controlled conditions and in this process polymer solution that is thermodynamically stable immediately separates into polymer-rich and polymer-lean phases . This teElectrospinning for the production of interconnected and continuous micro/nanofibers from a variety of materials is considered a universal technique . ElectroSeparation through a membrane usually takes place according to membrane morphology and is generally based upon different transport/permeation mechanisms. Main mechanisms of transport including Knudsen diffusion, solution diffusion and molecular sieving are proposed for gaseous transport across membranes usually depending upon the membrane matrix porosity  as shownA lot of research has been conducted to resolve the problems suffered by both inorganic and polymeric membranes. The deficiencies endured by both inorganic and polymeric membranes drove the researchers to create an advanced membrane material that possesses improved mechanical strength as well as higher separation performance and is also economically feasible. Mixed matrix membranes (MMM) which area combination of inorganic and organic materials were then put forward as an idea to achieve enhanced gas separation performance at a low cost. MMM fabrication is considered to be a favorable technology because this composite material enhances mechanical properties as well as amalgamates the superior separation characteristics and stability of inorganic material along with the efficient processability of polymeric material ,98. In M2 as it consists of Lewis basic sites which work as carriers of CO2 [The white part in the figure above exhibits the continuous phase of the polymeric matrix and the dispersed phase (fillers) is described by small, dotted squares. After the novel review on MMM by Okumus et al. , many res of CO2 . Therefos of CO2 . Many ty2/CH4 decreased up to 80% by the embodiment of unmodified zeolites due to unselective void formation [4/CO2 selectivity has boosted 50% as compared to neat membrane. By the absence of unselective voids after modification of the surface, CH4 followed a longer permeation path, whereas CO2 can easily access through the filler and hence selectivity of CH4/CO2 improved. Till now many reports have been patented on zeolitic-based MMM as a better alternative to polymeric and inorganic membranes [In the fabrication of MMM, solid polymer MMMs have received the most attention. In this type of MMM, both zeolitic and non-zeolitic inorganic fillers can be incorporated into the polymer matrix . Silico-ormation . Whereasembranes .2 plasticizes a broad range of glassy polymers [2 also causes an increase in polymer chain segmental mobility, and hence, increases diffusion coefficients of all the penetrants within the membrane. In order to improve plasticization resistance, modification and cross-linking methods were applied widely [2, long-term stability and gas separation performance can be achieved. To obtain a defect-free membrane with better separation performance, priming and sonication technique because of their simplicity is also adapted in the fabrication of MMM. The particle size of filler, besides sonication also helps in better filler dispersion throughout the polymer matrix. Filler particle size that is smaller than 50 nm gives more polymer filler interfacial area and hence enhances the interfacial contact between them [1/R2) and by (R1/R2)3 [During the process of gas separation, plasticization is another important phenomenon . At an epolymers . Reductid widely . The proeen them . Speakin(R1/R2)3 . Liu et (R1/R2)3  showed t(R1/R2)3 . Moreove(R1/R2)3 .2/CH4, and 43.5 barrer permeability of CO2, the resulting MMM with CMS exhibited favorable properties for separation. The improvement in separation properties was due to the incorporation of CMS into the polymer matrix.Non-zeolitic inorganic fillers have also received much attention in the development journey of MMM. Metal oxide nanoparticles, carbon molecular sieves (CMS), and porous and non-porous silica nanoparticles are some groups of non-zeolitic fillers. CMS nanoparticles having micro pores are incorporated in the fabrication of MMM as they exhibit remarkable permeation behavior and high productivity . They alIn order to fabricate heterogeneous Mixed Matrix membranes by a sol-gel process, porous and non-porous nanoparticles of silica in forms such as ceramic, tetraethoxysilane, organosilicate, fumed or colloidal silica is usually dispersed in the polymer matrix . BecauseA rapid increase in publications has been observed directing towards the applications of the above-mentioned fillers used in the fabrication of solid-polymer MMM. In spite of this, many alternative fillers such as layered silicates, graphene, carbon nanotubes (CNT), metal-organic frameworks (MOFs) have been studied as a new material for fillers having several attractive properties. The remarkable smoothness of the potential energy surface of CNT permits fast diffusion of gas molecules across their channels which in turn results in higher gas permeability without altering the selectivity. Some disadvantages of CNT that restricts its use in the MMM domain include high cost for production, inadequate adhesion between polymer matrix and particles of CNT, as well as entanglement and agglomeration of CNT particles. To improve the compatibility of CNT particles with polymer matrix, surface modifications that are carried out by acid treatments with coupling agents having hydroxyl or carboxyl group is considered to be a frequently used functionalization method. Acid treatment helps in better dispersion of CNT particles in polymer matrix and aids in higher gas molecular diffusivity by opening up the closed ends of CNT ,129. Gra3 (BTC)2 [Metal-organic frameworks (MOFs) have established a new approach towards the idea of solid fillers in the fabrication of MMM and have evolved as a key material for gas separation, storage and adsorption ,135. Thi3 (BTC)2  have pro2 [Zeolite imidazolate frameworks (ZIFs) belong to the class of MOFs and possess excellent thermal, chemical and hydrothermal stability up to 400 °C . One of 2 . In rece2  is a proWhen compared to the other applications, the applications of (MOFs) metal-organic frameworks for the separation of gases are still not very much developed. Despite the fact that MOFs possess many advantageous properties, it belongs to a new class of materials. Understanding of several factors including material cost as well as the influence of different vapors and gases is important prior to its use in industrial applications . In comp2 82.6 times more than their own volume [ZIFs belong to the class of Metal-Organic frameworks and are caged compounds formed by self-assembling of molecules, in which divalent cations, i.e., Zn or CO are coordinated tetrahedrally and are linked by immidazolate anions to form topologies that are similar to those of aluminosilicates zeolites . ZIFs han volume  due to t2 and H2 [2 affinity which is associated with the interaction among quadrupole moments of CO2 with open metal sites and polar functional groups in ZIFs [2 through a molecular sieving mechanism. Improved molecular exchange, as well as storage, is expected from the small aperture and large cages as compared to MOFs having a straight tubular channel.Because of the strong metal-ligand interaction, it is able to retain its structure even at high pressure and temperature owing to its hardness strength and high elastic modulus. The high physical density of ZIFs provides it stiffness to endure high loads hence enhancing its mechanical stability . ZIFs ar2 and H2 . Even at in ZIFs . ZIFs co3OH), N, N-diethylformamide (DEF) and N, N-dimethylformamide (DMF) in synthesis medium have resulted in being harmful to the environment because of toxicity present in their nature [2 capture, ZIFs have been researched broadly as membrane materials and adsorbents as they helps in reducing the rising level of CO2 in atmosphere caused from industrial emissions [Various methods, such as microwave-assisted solvothermal , solvothr nature ,156. To missions .+2 and organic linkers, as well as hydrophobic pores, are the reason behind its strong chemical stability [2/g and can be easily synthesized. Another remarkable property of ZIF-8 is its crystal size controllability. Its crystal size is mostly controlled by solvent type, synthesis temperature, rate of mixing, base type additive and the ratio of metal salt-ligand-solvent [ZIF-8 is one of the most explored Metal-Organic Frameworks. It possesses a porous crystalline structure having M-Im-M angle that is about 145° and usually coincides with Si-O-Si angle present in many zeolites having a large pore size of 11.6 A° and 6-ring window aperture of 3.4 A° as shown in tability . High thtability . Due to tability . ZIF-8′stability . ZIF-8 h-solvent . ZIF mat-solvent  in an aq-solvent . The pro-solvent ,191,192,-solvent  and cata-solvent .2 adsorption properties, it can be used as a membrane material as well as an adsorbent for purpose of gas separation [2 separation because of its crystallographic pore size which is 3.4 A° and it lies between the pores sizes of CH4 (3.8 A°), CO2 (3.3 A°) and N2 (3.64 A°). Because of these reasons, ZIF-8 is considered one of the most important filler materials in CO2 separation [The development of defect-free MMM is considered challenging as its fabrication suffers from polymer filler incompatibility. Poor interaction between polymer and filler that leads to the formation of non-selective voids can cause pore blockage, polymer rigidification and sieve-in-cage morphology, which deteriorates membrane performance . Thus, hparation ,195. ZIFparation . Second,paration . The filparation . A smallparation . Studies4/CO2, i.e., around 300% better than neat Matrimid membrane [4 was restricted. Interestingly, as per the author’s report, ZIF-8 agglomeration was observed even at 60 wt% loading, the selectivity of CH4/CO2 of the produced mixed matrix membrane was twice that in comparison to the neat membrane. Without the decline in the performance of the membrane, compatibility of Matrimid with ZIF-8 allowed higher loading of filler. As the loading of ZIF-8 increased up to 80 wt%, the membrane became fragile. Zhang et al. [2 and N2 atmosphere and ammonia impregnation can increase the amount of basic sites on the surfaces of samples and hence results in an improvement in selectivity and adsorption capacity towards CO2 [Studies conducted by Ordonez et al.  on ZIF-8membrane . The betg et al.  demonstrards CO2 .Porous solids having greater permeability as well as selectivity than polymers are mostly preferred as fillers in MMMs. As discussed earlier, many different filler particles including MOFs, zeolites, CMS, and other nanoparticles were incorporated into MMMs to enhance their gas separation performance. Interfacial interactions among these porous particles and polymer such as chain rigidification, pore blockage, and increase in free volume, as well as the formation of interfacial voids usually control gas transport through MMMs ,200.S) and diffusivity (D). In MMMs, the variations in permeability can be described by utilizing solubility and diffusion coefficient. The influence of polymer-free volume on the diffusion coefficient of penetrants can be explained by employing the statistical mechanical hard-sphere model of diffusion in liquids that were put forward by Cohen and Turnbull. The diffusion coefficients of penetrants (D) can be explained by employing the following Equation (1).Generally, permeability is described as the product of solubility ).According to the above equation, it is expected that an increase in free volume can enhance the diffusion of penetrants. The free volume that was measured by utilizing PALS (positron annihilation lifetime spectroscopy) in PTMSP/FS (Poly(1-trimethylsilyl-1-propyne)/Fumed silica) MMM displayed enhancement in free volume with the loading of FS and correspondingly substantial increase in permeability of Nng of FS ,202. TheHs = enthalpy of sorption; T = absolute temperature.ΔHs, and as a result the solubility of gas increases. Within poly(amide-6-b-ethylene oxide) and silica MMM, an increase in silica loading resulted in an increase in CO2 solubility coefficient. It was because of the strong interaction of molecules of CO2 with SiO2 as well as with polyimide block in PEBAX [An increase in interaction among penetrant molecules and functional groups decreases Δin PEBAX .Industrial applications involving membranes require thousands of square meters in order to execute a gas separation process at a large scale. Hence, the process of membrane separation should be efficient and economical if it is to be used commercially. Membrane configurations typically refer to the geometry of the membrane and its position in connection with the flow of feed and permeate. Moreover, it determines the pattern in which membranes are packed in modules. Membrane modules are actually a choice of configuration in which various formats including hollow fibers, spiral wound, plate and frame and tubular membrane modules are available. Hollow fiber (HF) membrane modules are preferred in industries because of their self-supporting ability, easy handling in module construction, larger surface area per unit volume as well as good gas separation abilities .The concept of hollow fiber (HF) membranes and their modules prepared from polymeric materials was first introduced by Mahon almost 50 years ago in his patents . Hollow 2/m3 [Chung and Kafchinski proposed that the formation of hollow fiber membrane is also controlled by rheological properties of spinning dope, the flow rates of bore fluid, properties of external coagulant, dope and bore fluid flow rates, temperature and also shear stress within spinnerets’ annular orifice . The par2/m3 . The tub2/m3 . HF memb2/m3 ,219,220.Membranes can be fabricated by stretching (porous membranes), nucleation-track-etching , melt-extrusion (dense membranes), particles-sintering (porous membrane), template-leaching (porous membranes), solution coating, phase inversion (asymmetric membranes) and swelling a dense film (porous membranes), etc. Most of the polymeric membranes are asymmetric, formed by the method of phase inversion and are available commercially. Many studies have been performed on the fabrication and characterization, as well as on the applications of HF membranes over the last few years and among them, polysulfone (PSF), poly vinylidene fluoride (PVDF), poly tetrafluoroethylene (PTFE), poly ethersulfone (PES), poly acrylonitrile (PAN) are membrane materials that are mostly used. When the material used to fabricate the membrane is selected, the mechanical strength and permeability of the membrane are determined by its structure, which mostly depends on the process and technique used to fabricate the membrane. Membrane’s mechanical stability especially long-term robustness and resistance towards chemical cleaning processes are some of the important factors alongside permeability and selectivity. HF membranes that are prepared by immersion precipitation technique normally have high permeability but low mechanical stability because of the loose support layer and dense layer. Therefore, hollow fiber membranes can easily get damaged or broken frequently in commercial applications by high pressure, frequent chemical cleaning, and airflow. Hence, HF membranes having exceptional mechanical strength and adequate separation properties are essentially needed.In order to increase the mechanical strength and properties of hollow fiber membranes, many studies have been performed. The use of high strength tubular braid that is coated with a separation layer is one of the techniques that is found to be effective . After tA membrane should have adequate mechanical strength, high permeability and good chemical stability. The chemical stability of the membrane is generally determined by the membrane material’s chemical composition. While, the permeability and mechanical strength of the membrane are dependent on the structure of the membrane, which is determined by the process used to fabricate the membrane . MechaniThe fabrication of reinforced hollow fiber membrane has not yet been extensively analyzed in research-based literature. Cooper et al.  first deThe phase inversion method is used for the fabrication of hollow fiber membrane. A typical production line of hollow fiber membrane is shown in HF membrane is categorized into reinforced type membrane and a single membrane type. In the reinforced-type of membrane, polymer resinous thin film is coated on a tube shaped braid or fabric while in single membrane type, hollow fiber membrane consists of polymer resin thin film without the utilization of supporting material such as tubular braid/fabrics. A support layer made of Polyethylene terephthalate is normally used as a braid support layer.The reinforced hollow fiber membrane shows excellent mechanical properties as it uses a tubular braid or fabric as a reinforcing material. One of the major problems in membrane processes is membrane fouling. Delamination is the membrane peeling off the surface of the braid and can occur due to cleaning of the membrane by back flushing. Out of many membrane materials utilized to fabricate hollow tubular reinforcing braids , braid composed of fiberglass is more prone to delamination because of poor adherence of membrane to the surface of braided material .The quality of the braided support layer can be determined by many parameters. Braided textile material has many breaks in fibers and is manufactured with standard equipment used for braiding and is made from yarn that is commercially purchasable. Fuzz, which is the build-up of broken fibers, can cause imperfections in polymer film that is coated on the braid’s surface. Whiskers which are splintered filaments stick out from the support layer’s surface and can result in polymer lean layers with pinholes or polymer layers of increased thickness.Braids such as hybrid, homogenous and heterogeneous are accessed by the interfacial bonding between the tubular braid and separation layer. A braid support hollow fiber membrane was fabricated by Lee et al.  in whichFan et al.  in anothTwo-dimensional braiding technique was used to prepare a hybrid braid consisting of PAN and CA filaments as depicted in In a study conducted by Quan et al. , homogenPotassium hydroxide) solution (3 wt%) for about 1 h at 90 °C or KOH solution (1 wt%) for about 6 h, the bonding strength between modified PET (1.1 MPa) braid and hydrophilic coating layer was two times greater than that was between original PET braid (0.6 Mpa) and the coating layer. Hence, this new approach is expected to enhance the bonding strength between the braid and the coating layer without changing membrane properties and it also has potential for operation in membrane engineering.In another study conducted by Zhou et al. , BRHF waIn a study conducted by Liu et al.  PVC BRHFDifferent types of braid are shown in If the support layer’s open weave has a very high porosity, polymer penetrates into the bore of the braid and there will be a sudden reduction in permeability. If the weaning is very tight, it will lead to poor adhesion of polymer and there are higher chances of peeling of the polymeric layer from the braid. The weaning of fiber is an essential parameter for the fabrication of membrane. Fibers that are weaved tightly can foul because of space restriction and, in turn, leads to insufficient moving in order to remain clean or rubbing against each other. On contrary, loose packing of fibers will help fibers in improved repetitive twisting. Cylindricity which is defined as fiber circularity is important. Cylindricity that is less than 0.8 caused uneven thickness of membrane that led to irregular flux as well as defective areas which ended in a film of polymer with undesirable thickness variations leading to variations in flux and defective areas that are rapidly fouled. The cylindricity of the braid should be close to 1.0 .Young’s modulus is another important braid parameter. The aim is to attain high strength by selecting a strong yarn such as aramid, glass and other materials with greater modulus to get an advantage from the material’s stability and high strength. For instance, a braid fabricated from threads of glass multi will have lesser than 5% elongation at break, moreover, these are also considered to be non-shrinkable. High modulus yarns usually impart inadequate film adhesion to the braid surface and are not desirable. If the braid is wet, it will be too weak for prolonged service .The concentration of polymer and additives along with their types regulate the structure of coated film layer. To fabricate the BRHF membrane in which a thin layer of film is coated onto the surface of supporting material or reinforcing material of the tubular braid, thermodynamic stability varies depending upon the dope solution composition that is used for coating. If the dope solution is thermodynamically stable then the predicted cross-sectional structure is mostly finger-like, and if the dope solution is unstable thermodynamically, then there will be no defected regions and the expected structure will be sponge-like.N-methylpyrrolidone (NMP) [The dope solution contains polymer, porogen (most probably a hydrophilic additive) and organic solvent for the polymer used. Typical polymer resins are polyethersulfone (PES), polyacrylonitrile (PAN), cellulose acetate (CA) , polyimine (NMP) , dimethyne (NMP) . Commonlne (NMP) , glycerone (NMP) .To fabricate the braid reinforced hollow fiber membrane Lee et al.  utilizedDuring the fabrication process of braid reinforced HF membrane, the coating of polymer is accomplished by using the unique design of spinneret. Generally, membrane thickness that is obtained outside of the braid is between 0.01 mm and 0.1 mm .The second nozzle design type that is utilized to fabricate the capillary membrane is shown in Another nozzle design is shown in To achieve the predetermined thickness of the separation layer on the reinforcing material’s surface, BRHF membrane’s spinning speed is crucial. The quantity of dope that is introduced in the spinneret, as well as the advancing speed of the tubular braid, must be balanced. The relation between the feed rate of dope solution and the tubular braid’s advancing speed is expressed by Lee et al. (2008) and is defined in Equation (3) .(3)Q=DoQ = Dope solution feed rate (ml/min); oD = braid outer diameter; T = dope solution thickness.As mentioned above, the relationship between the advancing speed of tubular braid and dope solution feed rate is revealed by Lee et al. . Accordi2. Greater values of K lead to the formation of a thick coating layer (Equation(4)) [The most appropriate coating is obtained when K is in the range of 200–300 g/mtion(4)) .(4)k, PET and PMIA/PET (1:1) braids were used by Chen et al.  to produChen et al., (2017) used BRHF membranes in MBR for the purpose of water filtration . The res2 removal from CH4 has highlighted different techniques of separation with their own disadvantages and advantages. Among all methods of gas separation such as absorption, adsorption and cryogenic separation, the membrane-based gas separation process is the most facile, environmentally friendly and simple process. Several spinning parameters were investigated for the fabrication of defect-free membrane. Moreover, according to previous research studies, nanoparticles of ZIFs due to their unique structures show superior adsorption capacity of CO2 as compared to the other MOFs.Previous research work about COSome of the advantages of HF membrane include a higher specific surface area along with lower requirements of maintenance and pre-treatment. However, high pressure can cause damage to HF membranes. Braid reinforced HF membranes provide a solution to the problem of mechanical strength faced by hollow fiber membranes. Few research studies are available in the open research literature on this subject. The main focus of these studies is on the utilization of polymer type, braid type, design of spinnerets as well the spinning speed of membrane. This review explains the research conducted on fabricating braid reinforced hollow fiber membranes in previous literature as a summary. One of the most important parameters for fabricating the BRHF membrane is good interfacial bonding between the separation layer and braided support. By selecting polymer types and braids that are compatible with one another, interfacial bonding can be improved. Reinforced HF membranes via employing braids can enhance the mechanical strength of membranes, hence, allowing separations at elevated pressures. Due to their high mechanical strength, they are used in UF membranes in the treatment of wastewater by MBR technology. The literature showed that the trend of the use of BRHF membrane increased in the last ten years. The trend also revealed that the use of reinforced membranes has been varied lately. These days, braid reinforced hollow fiber membranes can be utilized in MBR, UF, RO and NF processes. In the future, research should be performed on the use of braid reinforced hollow fiber membrane in gas separation areas especially for natural gas purification as they have high mechanical strength and it is expected that they can perform well in gas separation areas as they can handle high feed pressures. Moreover, further research should be conducted on the fabrication of braid reinforced HF membranes by employing other techniques such as grafting and blending.To date, rare studies are reported on the use of BRHF membrane incorporating ZIF-based filler for the purpose of gas separation. Hence, there is a need of conducting further research to analyze the improvement in performance while using BRHF membranes. Furthermore, in the future, the applications of braid reinforced HF membranes are foreseen in various different fields.


Our objective was to describe long COVID symptomatology according to initial coronavirus disease 2019 (COVID-19) severity.Predi-COVID cohort study participants, recruited at the time of acute COVID-19 infection, completed a detailed 12-month symptom and quality of life questionnaire. Frequencies and co-occurrences of symptoms were assessed.P < .001) and had on average 6.8 more symptoms  than initially asymptomatic participants who developed symptoms after the acute infection. Overall, 12.5% of the participants could not envisage coping with their symptoms in the long term. Frequently reported symptoms, such as neurological and cardiovascular symptoms, but also less frequent ones such as gastrointestinal symptoms, tended to cluster.Among the 289 participants who fully completed the 12-month questionnaire, 59.5% reported at least 1 symptom, with a median of 6 symptoms. Participants with an initial moderate or severe acute illness declared more frequently 1 or more symptoms (82.6% vs 38.6%, Frequencies and burden of symptoms present 12 months after acute COVID-19 infection increased with the severity of the acute illness. Long COVID likely consists of multiple subcategories rather than a single entity. This work will contribute to the better understanding of long COVID and to the definition of precision health strategies.NCT04380987. Since March 2020, the coronavirus disease 2019 (COVID-19) pandemic has disrupted the entire world population. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus infected >315 million people and caused >5.5 million deaths worldwide as of January 2022 [Indeed, a fraction of the people with COVID-19 experience continuing effects of the disease, with complaints such as tachycardia, extreme fatigue, and inability to perform daily physical tasks . The sigLong COVID can affect adults and children and many of them did not return to the same level of work and quality of life as before COVID-19 infection .Both hospitalized and nonhospitalized persons during acute infection are likely to develop long COVID. However, it remains unclear to what extent the persistence of symptoms or the appearance of new symptoms is related or not related to the severity of acute illness , 10.An increasing number of studies report long-term health consequences in asymptomatic or mildly symptomatic patients or hospitalized patients or in mixed cohorts with COVID-19 .For all of these reasons, the identification of predictive markers and risk factors of the long-term sequelae of COVID-19 has been defined as a research priority .Our hypothesis is that long COVID symptomatology may differ according to the initial COVID-19 disease severity and that symptoms can cluster and define subtypes of long COVID. To test it, we aimed to (1) provide an overview of the symptoms reported 12 months after the acute infection among a cohort of COVID-19–positive adults in Luxembourg, composed of study participants with various forms of disease severity during the acute illness; (2) perform a co-occurrence analysis of the symptoms; and (3) evaluate the impact of long COVID on quality of life.Data were obtained from participants from the Predi-COVID study, a prospective hybrid cohort study of persons with a polymerase chain reaction (PCR)–confirmed diagnosis of COVID-19 in Luxembourg. The Predi-COVID study design and analysis plan has been published previously . In brieBaseline characteristics were collected at study inclusion, which was performed in the 5 days after the PCR test result. Participants were then followed digitally 12 months after inclusion with self-reported questionnaires on symptoms and quality of life.The written consent of all participants was obtained before inclusion in the study. Data collection in Predi-COVID follows the best practices guidelines from the German Society of Epidemiology . Predi-CThis study is an analysis of participants’ symptoms and health status 12 months after the acute infection. All participants included between 1 May and 8 November 2020 were eligible for the present study (N = 539) and were invited to complete a detailed 12-month questionnaire. Among them, 330 completed the questionnaire (response rate of 61.2%). We further excluded 41 participants with incomplete data and the final study population was composed of 289 participants. Characteristics of the respondents were compared to those of the nonrespondents .The detailed 12-month questionnaire was inspired from that co-developed by Tran et al  and consParticipants were also requested to answer the question “Have you noticed the following symptoms or illnesses since your COVID-19 diagnosis?” Response modalities were: 1, “Yes, and I still feel it today”; 2, “Yes, I had it but I no longer have it”; and 3, “No, I have never had this symptom.” A variable “total number of symptoms” has been created that corresponds to the sum of all symptoms still present at 12 months.Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI) questionnaire. A categorical variable was generated using the PSQI score: poor sleep was defined as PSQI total score >5 .The respiratory quality of life was assessed using the VQ11 questionnaire. One global score and 3 subscores  were calculated as described elsewhere , 22 and Finally participants were asked if they could manage their current state of health in the long run, taking into account all the symptoms that they have experienced in the last 30 days in terms of frequency, intensity, and impact on their life and that can be attributed to COVID-19 (yes/no).Our analysis took into account the following set of covariates: age, sex, body mass index, smoking status , and comorbidities . The disease severity at inclusion was also used as a potential determinant of developing a more severe form of long COVID.Disease severity during the acute phase of the disease was defined in 3 categories according to an adapted version of the National Institutes of Health severity classification : asymptot test and 1-way analysis of variance to determine the differences of distribution for continuous variables and Fisher exact test for categorical variables. Kruskal-Wallis test was used to determine the differences for the variable total number of symptoms.We described the continuous variables, which were normally distributed, as mean ± standard deviation, and categorical variables as numbers (percentage). We used the Student We studied the co-occurrences of long COVID symptoms at 12 months and clustered the symptoms using Ward’s hierarchical clustering method with Euclidean distance .We performed all of the analysis using the R language  and geneP = .029; Individual characteristics of respondents and nonrespondents to the 12-month questionnaire were similar except for sex, with women being slightly overrepresented in the study population  participants presented at least 1 persisting symptom 12 months after acute infection. Among them, the median total number of persisting symptoms was 6  and one-third experienced >10 symptoms .Four of the 64 symptoms were not reported by any participant . The most frequently reported symptoms were fatigue (34.3%), eye tiredness (22.5%), shortness of breath (18.7%), and irritability (18.0%) .P < .001), and many symptom frequencies were increased  and had a more altered respiratory quality of life  when compared to asymptomatic participants.Poor sleep was reported by 155 (54.2%) participants and 37 (12.9%) participants had an altered respiratory quality of life 12 months after the acute infection. The physical autonomy, psychological, and relational components of the respiratory quality of life score were affected in 7.3%, 12.9%, and 3.9% of the participants, respectively. Participants with a moderate/severe form of the disease during the acute phase reported more frequently poor sleep  answered “No” to the question on whether they could manage their current health status in the long run . This reOur study provides a comprehensive overview of the symptoms reported by people with long COVID 12 months after an acute SARS-CoV-2 infection. We found that almost 60% of the participants experienced at least 1 symptom, with a predominance of fatigue, dyspnea, and anxiety. The presence of symptoms at 12 months was associated with a moderate or severe form of the acute infection. We also observed clusters of symptoms frequently co-occurring and showed that long-term symptoms had a high impact on quality of life.In our cohort 59.5% of the participants who fully completed the 12-month questionnaire declared at least 1 symptom 1 year after the acute infection. This proportion was higher for participants with moderate/severe form of acute illness (82.8%) than for participants with mild or asymptomatic initial form of the illness . Other prospective cohort studies report similar frequencies of participants experiencing symptoms 12 months after acute infection , 29.We obtained similar patterns as reported in other studies, with a variety of symptoms and a predominance of fatigue, dyspnea, and anxiety , 30–32. Several mechanisms can explain this wide panel of symptoms: SARS-CoV-2 virus can enter cells of many organs via the ACE2 receptors and thus provoke multiorgan damage. Chronic fatigue can be explained by a dysfunction in inflammatory response pathways, but other factors may be involved too. Neurological and cognitive impairments may be explained by chronic neuronal inflammation and damage since SARS-CoV-2 is able to pass the blood-brain barrier . An acutThe total number of symptoms present 12 months after acute infection is difficult to compare with existing studies as the cohort types, the timepoints, and the way to collect symptoms are different , 10, 35.Participants with a moderate or severe form of acute illness had a significantly higher propensity to declare symptoms 12 months later and had a more altered quality of life than those with asymptomatic or mild form of acute illness. Previous studies reported contradictory findings as some studies did not find an association between long COVID and initial disease severity during acute COVID-19 , but othWe observed several clusters of symptoms that often co-occurred. Among frequently reported symptoms, we observed a distinct cluster mainly constituted by neurological symptoms and another by pain-related symptoms. Cardiovascular symptoms were present in both clusters but did not constitute a separate cluster. In less frequently reported symptoms, we have seen that gastrointestinal symptoms  on one hand and loss of taste and loss of smell on the other hand usually co-occurred. This is consistent with findings from other studies confirming that different clusters of long COVID can be described and could benefit from adapted treatments and diagnosis , 42.Symptoms present at 12 months had an important impact on participants’ quality of life, with sleep disturbances, altered respiratory quality of life, and discouragement concerning their state of health. People who experienced a moderate or severe form of initial COVID-19 were the most impacted in terms of sleep and respiratory quality of life. Participants who declared not being able to cope with their symptoms in the long run had similar individual characteristics than those who declared the opposite, and had reported higher symptom frequencies. This further underlines the impact of long-term symptoms on quality of life.Our study has several strengths. First, it is a prospective cohort study including COVID-19 positive persons combining various acute illness stages and followed 12 months after acute infection compared to other studies . All stuThis study also has several limitations. The response rate to the long COVID questionnaire was 61.2%, which could lead to an overestimation of symptoms since people without any symptoms might have been less inclined to complete the questionnaire. Participants who responded to the questionnaire had the same individual characteristics as participants who did not respond, which limits but does not completely rule out the risk of selection bias, which could then lead to an overestimation of some symptom frequencies.since your COVID-19 diagnosis?” limits the risk of reporting symptoms not related to COVID-19. Moreover, the appearance of symptoms in asymptomatic patients or after recovery of the acute illness has been reported by other studies [We cannot fully state that all symptoms reported in the 12-month questionnaire are associated with COVID-19, in particular for newly appeared symptoms in participants asymptomatic during the acute phase of the infection. However the wording of the question “Have you noticed the following symptoms or illnesses  studies , 45. It  studies .Symptom intensity was not assessed, except for sleep quality and respiratory quality of life, for which we used validated scales. We did not have the information on potential medication taken by the participants to reduce their symptoms, and this could lead to an underestimation of some symptoms.Study participants were included before the COVID-19 vaccination campaign started in Luxembourg and thus were not vaccinated at inclusion, with unknown vaccination status after 12 months of follow-up. Therefore, we could not assess the impact of COVID-19 vaccination on the development of long COVID. This would be of high interest to replicate our study as some studies show an association between vaccination and a lower long COVID risk, whereas others have shown no association , 48.Our study provides an extensive description of symptoms present 12 months after COVID-19 infection and their impact on the quality of life of COVID-19 patients in a well-characterized prospective cohort. We highlighted a significant burden for people living with long COVID 12 months after the initial infection. An initial moderate or severe illness was associated with an increase in long-term consequences of COVID-19. Finally, our study helps to define long COVID and confirms that it is multisystemic and presents different clusters of symptoms. These results will ultimately help to better identify long COVID in clinical settings and contribute to the definition of precision health strategies.ofac397_Supplementary_DataClick here for additional data file.


Sexual dysfunction can be caused by impaired neurotransmission from the peripheral to the central nervous system. Therefore, it is important to evaluate the input of sensory information from the peripheral genital area and investigate the control mechanisms in the spinal cord to clarify the pathological basis of sensory abnormalities in the genital area. However, an in vivo evaluation system for the spinal cord–penile neurotransmission mechanism has not yet been developed. Here, urethane-anesthetized rats were used to evaluate neuronal firing induced by innocuous or nociceptive stimulation of the penis using extracellular recording or patch-clamp techniques in the lumbosacral spinal dorsal horn and electrophysiological evaluation in the peripheral pelvic nerves. As a result, innocuous and nociceptive stimuli-evoked neuronal firing was successfully recorded in the deep and superficial spinal dorsal horns, respectively. The innocuous stimuli-evoked nerve firing was also recorded in the pelvic nerve. These firings were suppressed by lidocaine. To the best of our knowledge, this is the first report of a successful quantitative evaluation of penile stimuli-evoked neuronal firing. This method is not only useful for analyzing the pathological basis of spinal cord–penile neurotransmission in sexual dysfunction but also provides a useful evaluation system in the search for new treatments. Peripheral nerve dysfunction is associated with several sexual dysfunctional disorders  ,2,3,4,5.Therefore, it is important to evaluate the input of sensory information from peripheral regions, such as the genital area, and the control mechanism in the spinal cord to clarify the pathophysiological basis of sensory abnormalities and pain in the genital area. In addition, inhibition of peripheral nerve firing is promising in the development of therapeutic drugs for sexual dysfunction and genital sensory disturbance , and an Genital stimuli are input to the dorsal horn of the lumbosacral spinal cord (L6–S1) via peripheral nerves ,11,12,13In this study, we developed a novel method to evaluate neuronal firing in the spinal cord and peripheral nerves following genital stimulation in rats. For the spinal cord, we developed a method to evaluate neuronal firing when the genital region was stimulated using extracellular electrophysiology in the dorsal horn of the spinal cord in the lumbosacral region or the patch-clamp method. For the peripheral nerves, we also developed a method to evaluate nerve firing in the pelvic nerve during mechanical stimulation of the genital area.We attempted to evaluate evoked neuronal firing in response to penile stimulation. To record the data, we constructed a novel system for in vivo electrophysiological evaluation, including extracellular recordings and patch-clamp recordings at the dorsal horn of the spinal cord in the lumbosacral region (L6–S1), which receives input from the genital area A,B. ThisWe conducted extracellular recordings of neuronal firing in the deep spinal dorsal horn (lamina III and IV) of the spinal cord during innocuous mechanical stimulation equivalent to tactile stimulation of the genital area A.First, to identify neurons that respond to penile stimulation and their receptive fields, the penis was subjected to innocuous stimulation using a brush, and the electrode position was fixed while recording the neuronal firing evoked by the mechanical stimulation B.Next, we used two different intensities of von Frey filament (vFF) (1.0 and 4.0 g) and evaluated the neuronal firing during each stimulus. We successfully recorded stimulus-evoked firing at all vFF intensities C. The fiAs described above, our method could evaluate stimulus intensity-dependent neuronal firing in the deep dorsal horn of the spinal cord in response to innocuous mechanical stimuli, such as touch to the penile region, and measure spontaneous firing.We conducted extracellular recordings of neuronal firing in the superficial spinal dorsal horn (lamina I and II) of the spinal cord during nociceptive stimulation of the genital area A.As previously described in the study of the deep spinal dorsal horn, we identified neurons that respond to penile stimulation and their receptive fields by recording neuronal firing during nociceptive stimulation of the penis using forceps B.Next, we used different intensities of vFF (4.0 and 26.0 g) and evaluated the neuronal firing during each stimulus. vFF stimuli-evoked firing was successfully recorded at all intensities C. The 4.To investigate the response to chemical stimuli, capsaicin (1%) was applied to the penis as a chemical stimulant, and the evoked neuronal firing was recorded  during extracellular recording in the deep spinal dorsal horn. These vFF-evoked neuronal firings were completely suppressed by perfusion of the spinal cord surface with lidocaine (3 mM) A.Next, neuronal firing was also recorded when the penis was stimulated with vFF (4.0 and 26.0 g) during extracellular recording in the superficial spinal dorsal horn. These vFF-evoked neuronal firings were also inhibited by perfusion of the spinal cord surface with lidocaine (3 mM) B. After We investigated the possibility of using the patch-clamp method to measure neuronal firing during penile stimulation in the superficial spinal dorsal horn in the lumbosacral region. Excitatory synaptic responses were successfully recorded, and stable recordings were acquired from 14 neurons (20–120 μm depth) , which receives input from the limbs ,19,20,21Peripheral neuropathy is known to be associated with some sexual dysfunctions ,2,3,4,5.3 receptors in response to nerve activity during penile stimulation as well as for behavioral evaluation [In this study, normal rats were used to develop a new experimental method, but it should be possible to use various pathological models for analysis. This method is also expected to be applied to mice  and can aluation .We successfully recorded spontaneous excitatory postsynaptic currents (sEPSCs) or vFF-evoked EPSCs in the superficial spinal dorsal horn, including both extracellular and patch-clamp recordings. However, further studies on the patch-clamp recordings are needed. For example, if we can analyze not only EPSCs but also inhibitory postsynaptic currents (IPSCs) for excitatory and inhibitory signals, a detailed analysis of synaptic responses will be possible, which may contribute to a detailed investigation of the molecular mechanisms that cause sexual dysfunction.Since in vivo electrophysiological evaluation in the spinal dorsal horn requires skilled techniques, we also investigated a brief evaluation method using peripheral pelvic nerves. This simple method is expected to be applied to screening evaluations in drug discovery research for peripheral sensory abnormalities in sexual dysfunction diseases. To investigate the findings of this method in detail, it is possible to take a multifaceted approach, such as using in vivo electrophysiological studies in the spinal dorsal horn.Wistar rats  were used for the electrophysiological tests in the spinal dorsal horn performed at the Toyama University. Wistar–Imamichi rats  were used for the electrophysiological tests at the pelvic nerve conducted at the Mitsubishi Tanabe Pharma Corporation (MTPC).All rats were housed under a controlled 12/12-light cycle (lights were on from 7:00 A.M. to 7:00 P.M.), temperature was 23 ± 3 °C, and humidity was 30–70%, with access to food and water ad libitum.All in vivo experimental procedures were performed in accordance with the “Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences” of the Physiological Society of Japan and were approved by the Institutional Animal Care and Use Committee of Research Laboratories at the University of Toyama and MTPC. All efforts were made to minimize animal stress and the number of animals used.2–5% CO2-balanced Krebs solution (8–12 mL/min) at 37 ± 1 °C. In vivo extracellular recordings were performed from the lumbosacral region (L6–S1), which receives input from the penile region (lower abdomen). Single-unit recordings of the deep spinal dorsal horn  or the superficial spinal dorsal horn  neurons were performed. Next, the spikes were selected for amplitude identification. A tungsten microelectrode  was inserted into the ipsilateral spinal cord at an angle of 20–30°  and recorded from neurons 180 to 400 μm below the surface (corresponding to lamina III and IV) or 15 to 150 μm below the surface (corresponding to lamina I and II). The unit signals were amplified , digitized , and displayed online using a special software package . We searched for areas on the skin where neurons responded to a light brush touch or pinching with tweezers. Mechanical stimulation was applied to the penile region for 10 s using a von Frey filament (vFF).Recordings of the adult rat spinal dorsal horn neurons were performed using a modification of previous methods ,21,22. ACapsaicin  was dissolved in ethanol, and the solution was applied to the penis. Lidocaine  was dissolved in Krebs solution and applied while perfusing through a three-way stop-cork.2 0.5, MgCl2 2, EGTA 5, HEPES 5, and ATP-Mg 5 (pH 7.2). The potassium gluconate solution was used during the recordings of spontaneous excitatory postsynaptic currents (sEPSCs) at a holding potential of −70 mV. The resistance at the tips of the patch pipettes was 6–12 MΩ. Using a micromanipulator , the electrode was advanced at an angle of 30 from the window into the lamina I–II and formed a gigaohm seal with a cell at a depth of 20–120 μm from the contact point to the dorsal surface of the spinal cord. Series resistance was evaluated based on the response to a 5-mV hyperpolarizing step. This value was monitored during the recording session, and data were rejected if values changed by more than 15%. Signals were obtained using a patch-clamp amplifier . The data were digitized using an analog-to-digital converter , stored using a data acquisition program , and analyzed with the software package . To record EPSCs, cell recordings were performed in voltage-clamp mode at holding potentials of −70 mV [Blind whole-cell voltage-clamp recordings were obtained as in previous methods ,21,22,26f −70 mV ,21,22.All rats were anesthetized with urethane . After fixation in the supine position, a median incision was made in the skin and muscles from the lower abdomen to the lower diaphragm. The surrounding connective tissue was dissected to expose the pelvic nerves. Pelvic nerves were placed on a silver–silver chloride bipolar electrode connected to a bioelectric amplifier  in an electric shield, and then, neuronal firing activity was recorded. Micro1401-3  was used as the interface. The nerve firings to be evaluated were defined as those evoked by stimulation of the genital area around the penis with a brush, and an average of two or more responses to 10 s of stimulation was used. Data were analyzed using Spike 2 software (Cambridge Electronic Design).Lidocaine  was dissolved in saline, and the solution was applied directly to the pelvic nerve.t-test. Differences were considered significant at p < 0.05. All statistical analyses were performed using the SAS software . In this study, n refers to the number of neurons (in vivo extracellular recording and patch-clamp recording) or animals .Data are presented as the mean ± standard error of the mean (S.E.M.). For evaluation in the pelvic nerve, nerve firing before applying lidocaine was set to 100% and compared to change after administration. For statistical analyses, we performed a one-way analysis of variance (ANOVA), followed by a two-tailed Wilcoxon rank sum test and a Student’s In this study, using in vivo extracellular recording, patch-clamp recording, and electrophysiological evaluation methods in peripheral nerves, we succeeded in recording penile stimulation-induced neuronal firing and synaptic current input to the spinal dorsal horn in the lumbosacral region via peripheral nerves. This is the first report on a methodology for the quantitative evaluation of neuronal activity during penile stimulation. Using this method, it is also possible to record and analyze the frequency of neuronal firing and synaptic currents in response to various types of nociceptive and innocuous stimuli. In addition, pharmacological studies using this method can be administered in the same manner and dosage as different behavioral pharmacological studies. Therefore, this method can be a useful evaluation system not only for analyzing the pathological basis of spinal cord–penile neurotransmission mechanisms in sexual dysfunction, which have not been well studied, but also for searching for new methods of treatment as well as therapeutic drugs.


The barrier function of the skin is mainly assured byits outermostlayer, stratum corneum (SC). One key aspect in predicting dermal drugdelivery and in safety assessment of skin exposure to chemicals isthe need to determine the amount of chemical that is taken up intothe SC. We here present a strategy that allows for direct measuresof the amount of various solid chemicals that can be dissolved inthe SC in any environmental relative humidity (RH). A main advantageof the presented method is that it distinguishes between moleculesthat are dissolved within the SC and molecules that are not dissolvedbut might be present at, for example, the skin surface. In addition,the method allows for studies of uptake of hydrophobic chemicals withoutthe need to use organic solvents. The strategy relies on the differencesin the molecular properties of the added molecules in the dissolvedand the excess states, employing detection methods that act as a dynamicfilter to spot only one of the fractions, either the dissolved moleculesor the excess solid molecules. By measuring the solubility in SC anddelipidized SC at the same RHs, the same method can be used to estimatethe distribution of the added chemical between the extracellular lipidsand corneocytes at different hydration conditions. The solubilityin porcine SC is shown to vary with hydration, which has implicationsfor the molecular uptake and transport across the skin. The findingshighlight the importance of assessing the chemical uptake at hydrationconditions relevant to the specific applications. The methodologypresented in this study can also be generalized to study the solubilityand partitioning of chemicals in other heterogeneous materials withcomplex composition and structure. To evaluatethe absorption and transport in the SC, it is crucial to know theoverall solubility of the chemicals of interest in the complex andheterogeneous SC material. In addition, to get insight into the distributionbetween different regions of the SC as well as what the transportroute across the SC is, one needs to know how the added chemical distributesbetween the extracellular lipids and the corneocytes.The outermost layer ofthe epidermis, the stratum corneum (SC),is composed of layers of corneocytes embedded in a lipid matrix withlamellar arrangement.5 A fundamental complication with these methods isthat besides the uptake of various chemicals from the formulationinto SC, there will also be extraction of molecules from the SC intothe external solution  ofthe environment. This illustrates the need for methodologies thatenable the quantification of solubility of various chemicals insideSC under ambient conditions and not only at full hydration or in exposureto other solvents.One important aspect to consider when characterizingsolubilityin SC is the hydration condition. Most of the existing protocols usedto measure the solubility of chemicals in SC are performed in conditionswhere the SC is placed in contact with an excess solution that containsthe molecule of interest.19 We here demonstrate the use of the method in studiesof the solubility of a range of chemicals representing different molecularclasses with respect to structure and hydrophobicity , 2-methyl resorcinol (2-MR), and 7-ethoxycoumarin were purchasedfrom Sigma-Aldrich. Metronidazole was from Duchefa Biochemie (Netherlands).Caffeine, phenylethyl resorcinol, and minoxidil were obtained fromL’Oreal (France). Phosphate-buffered saline (PBS) contained130.9 mM NaCl, 5.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4. Milli-Q water  wasused to prepare PBS and saturated solutions of caffeine and 2-MR.NaCl, Na2.2Porcine ears were obtained from a localabattoir as byproduct of food production and stored at −80°C until use. Hair was removed by a trimmer, and the skin fromthe inner ear was dermatomed  toa thickness of approximately 500 μm. To separate SC from tissue,the dermatomed skin strips were placed on filter paper soaked in PBSsolution with 0.2 wt % trypsin at 4 °C overnight. Sheets of SCwere removed by forceps, washed with PBS five times, dried under vacuum,and stored in a freezer until further use.20 using batches of smaller size (ca. 15 pig ears) that 13C NMR signals and molecular mobility of SC components are similarbetween the batches, confirming that the batch-to-batch variationis remarkably small. Additional WAXS experiments were performed onsamples composed of SC sheets and 2-MR prepared and equilibrated inthe same way as pulverized SC samples , showing no significant differences in the measured solubilityin the SC sheets and pulverized SC  was prepared, and all internal comparisons were performed withsamples from the very same batch of pulverized SC for all NMR (nuclearmagnetic resonance) and WAXS (wide-angle X-ray scattering) experiments.It is noted from comparisons between previous NMR studies on SC21 In short, the SC sheets were made into smallpieces and the extracellular nonbound lipids were extracted by threesolutions with different chloroform:methanol mixtures of ratios 2:1,1:1, and 1:2 v/v. This sequence of extractions was performed at roomtemperature under gentle shaking for about 2 h. The remaining SC materialwas collected by filtration after each step. The whole extractionsequence with all three solvent mixtures was repeated for about 30min for each extraction step. The filtered SC material was soakedin methanol overnight. In the final step, the methanol-extracted delipidizedSC material was rinsed in Milli-Q water several times and dried undervacuum in a desiccator. During the extraction, the SC small piecescan be fragmented and after the extraction, we obtained a batch containingof very small grains of delipidized SC. The delipidized SC obtainedfrom this procedure still contains covalently bound lipids in thecornified envelopes of the corneocytes.22 All experiments on delipidized SC were performedwith samples from the same batch. It is noted that any lipid extractionprotocol using apolar solvents may cause extraction of unwanted hydrophobiccomponents and slightly polar compounds from the sample. Still, previousstudies6 showed that soaking delipidizedSC that was prepared with this protocol in water leads to furtherextraction of NMF, indicating that the preparation procedure usingorganic solvents does not remove all NMF from the sample. It is alsopointed out that the molecular dynamics of the keratin protein andcorneocyte envelope lipids are very similar for the corneocytes insidethe SC sample and inside the delipidized SC prepared using the presentprotocol for a range of hydration conditions,10 implying that the extraction procedure does not change overall propertiesof the corneocytes significantly.Delipidized SC was prepared from another batch ofSC sheets (ca.40 pig ears) according to previously published protocols.2.32O. The samples and chemicals were added to the tube according tothis order: the solid added chemical, then the SC (delipidized SC)powder, and finally D2O. Using this preparation procedure,almost all of the added D2O is first absorbed by the SC(delipidized SC) samples, and therefore the risk of the solid chemicalbeing dissolved in D2O as a separate phase outside SC (delipidizedSC) during the mixing is very low. The samples with known compositionwere then centrifuged, mixed, and equilibrated at 32 °C in closedchambers for 1 day with an RH of 93 and 97% (D2O), controlledwith saturated solutions of KNO3 and K2SO4, respectively. The centrifugation step is to collect samplesor chemicals that could otherwise stick to the walls of the Eppendorftube and also helps the water absorption into SC (delipidized SC)samples as well as the mixing process to avoid the static in the powder.The water was never added in excess amount, and the RHs are alwaysbelow 100%, meaning that there is no excess liquid water in the presentsamples. The final D2O water content of each individualSC or delipidized SC sample at a certain RH was determined by gravimetricmeasures before and after equilibration using a Sartorius R160D balancewith a readability of 0.01 mg. Replicates of water content measurementstherefore were performed for samples with the same composition thatwere measured by both NMR and WAXS. The samples were then transferredinto tight rotor inserts to be fitted into a Bruker 4 mm rotor forNMR experiments or into a screw-tight sandwich cell, where the sampleis contained between two polyimide film (Kapton) sheets for WAXS measurements.Approximately 10–20 mg of dry pulverized SC or small grainsof delipidized SC was added to an Eppendorf tube together with a controlled amount of added chemical and D2O addedto the samples is almost the same as the D2O content determined fromthe gravimetric measures after 1 day for most samples, indicatingthat one has reached (or very close to) the equilibrium between watervapor phase in the RH chambers and water inside the samples. In previousstudies,20 SC samples were prepared almost in the sameway, i.e., mixing a certain amount of water and added chemicals withSC and then incubating the sample. No significant difference in 13C NMR signals and molecular mobility of both SC componentsand added chemicals between the samples incubated for 1 and 2 dayswas observed.7 One day is therefore consideredlong enough to reach equilibrium with respect to the water uptake.The absorption kinetics for the uptake of the added chemicals areexpected to strongly depend on the grain size of the particles. Oneway to confirm that the sample is not in the absorption kinetic controlledlimit is to vary the particle size. Here we studied the solubilityof 2-MR in both pulverized SC and SC sheets after 1 day of incubation, and the measured values werevery similar. If the uptake was controlled by absorption kinetics,we would expect a large difference between the solubility in the sampleswith small grains and larger sheets. With respect to uptake of theadded chemical, the absorption kinetics was studied for one sample(2% 2-MR in SC at 93%RH), showing that saturation was reached within10 min , justifying that wereached equilibrium with respect to the added chemical for this sampleafter 24 h. For the remaining samples, we did not study adsorptionkinetics, but still judge that 24 h equilibration is sufficient basedon the consistency within the full set of NMR and WAXS data for allof the systems investigated  and addinga preliminary amount of water that is estimated to be close to theequilibrium water content were to reduce the equilibration time, astoo long incubation times may cause problems with degradation of thebiological SC sample. The preliminary amount of D2.4In thepresent experimental approach, instead of performing several replicatesfor one sample prepared at one concentration, we studied a seriesof conditions  for each system. From the set of experiments performedfor each chemical, we can confirm consistency within the whole experiment,in the NMR case, a linear increase until saturation is reached. Inaddition, we study each sample with two different experimental methods(NMR and WAXS), again to confirm internal consistency within the datasets for each system. The series of NMR data were analyzed throughcurve fitting, and from the fit of all data points we obtain the concentrationat saturation. In case one of the data points is off, it would notstrongly affect the overall outcome from the fit. The variabilityobtained from the fitting therefore includes variabilities of bothsample preparation and the measurements, which are similar to thevariability that would be obtained from replicated measurements ofsamples with the same composition. The biggest variability in themeasurement of chemical uptake into SC is likely from the sample variationof the biological sample. This variability is minimized in this studyby preparing a batch containing SC from several individuals, and allof the samples in one solubility measurement are from the same batch.2.51HNMR experiments were performed on a Bruker Avance AVII-500 MHz NMRspectrometer (Bruker) with a Bruker 4 mm 1H/13C/31P HR (high resolution) MAS (magic-angle spinning)probe equipped with a MAS gradient coil and carried out at a spinningfrequency of 5 kHz and at 32 °C. Free induction decays (FIDs)of 1H NMR measurements were recorded after a 10 μs90° pulse using a spectral width of 20 ppm, an acquisition timeof 0.05 s, 8 scans per experiment, a recycle delay of 20 s, and areceiver dead time of 4.5 μs. The FIDs were Fourier transformedwith a line broadening of 10 Hz to obtain frequency domain NMR spectra.The 1H chemical shift of the terminal methyl peak at 0.9ppm in SC and delipidized SC samples was used as an internal reference,while for the other samples, the calibration from previous experimentswas kept. The following conditions were used to fit the data to findthe value of mAC-SC-sat/mSC corresponding to the saturated concentrationmAC-SC is the weight of the added chemical AC in the SC and mSC is the dry weight of SC. The Monte Carlo error estimationmethod23 was used to estimate 1000 valuesof mAC-SC-sat/mSC and its mean and 95% confidence intervals using a standardnormal distribution of the sample. The same procedure was used todetermine the saturation concentration in the delipidized SC .All 2.6q is defined as q = (4π sin θ)/λ, where θ ishalf of the scattering angle and λ = 1.54 Å is the X-raywavelength. Silver behenate which generates well-defined peaks ina q range of 0.1076 to 1.387 Å–1 was used as a reference to calibrate sample-to-detector distanceand detector positions.24 The two-dimensionalscattering pattern was radially averaged using SAXSGui software toobtain one-dimensional data without background correction.WAXS experiments were performedusing a SAXLab’s GANESHA 300 XL SAXS system equipped with a microfocus sealed tube X-ray source (GE’s InspectionTechnology) and a 2D 300 K Pilatus Solid State detector  at 32 °C. The temperature was controlled using aJulabo T Controller CF41 from Julabo Labortechnik GmbH (Germany).The scattering vector 31H MAS NMR25 and WAXS.26 We start by describing the results obtainedfor two model chemicals with different hydrophobicity, caffeine and2-methyl resorcinol (2-MR). Caffeine is a cosmetics ingredient andis a standard model chemical used for skin absorption assays.27 2-MR is a moderate skin sensitizer which canbe found in hair dye formulations.28 Wefirst measure the solubility of these chemicals in SC and in the delipidizedSC at different water contents. Thereafter, we broaden the scope andpresent experimental data of solubility in SC for a range of otherchemicals with different molecular structures and properties to leave time for the added chemical to dissolve in the SC matrixand the water content to adjust to a value corresponding to the chemicalpotential given by the RH of the environment. We then study thesesamples by means of 1H NMR and WAXS to detect the fractionof the added chemical that is dissolved in SC as well as the excessundissolved solid fraction. In the 1H NMR spectrum, solidcomponents are observed as a very broad peak, whereas dissolved componentsthat possess much faster molecular motion give rise to sharp peaks caffeine. We therefore conclude that the saturationconcentration of caffeine in SC at 97% RH obtained from WAXS is around3–4 wt % which is close to the values obtained from the NMRmeasurements. From the combination of the 1H NMR and WAXSexperiments, we are thus able to pinpoint the solubility of caffeinein the hydrated SC, and the data are summarized in Next, the samples composed of SC and caffeine at97% RH were examinedusing WAXS. 3.21H NMR spectra obtained at RH = 97%. Thedifferent resonances of the neat chemical are detected as broad peaksat this RH. When dissolved in SC at the same RH, one can detect sharppeaks of 2-MR in SC. To analyze these data, we integrate the peakfrom hydrogen 3 of 2-MR . The saturation concentrations of both chemicals decreasewith decreasing RH, both with respect to the total mass and the drymass of the SC samples . Metronidazole has antibacterial propertiesand is used in topical formulations for, e.g., the treatment of thechronic skin disease rosacea.31 Minoxidilis used to stimulate hair growth and treat androgenetic alopecia.32 7-ethoxycoumarin belonging to a coumarin familythat is widely used in cosmetic products is also used for metabolismcharacterization.33 And finally, phenylethylresorcinol is a skincare and cosmetic ingredient due to its skin-lighteningeffect.34 As seen in FSC,sat < 2 wt %) is seen for metronidazole, minoxidil,and 7-ethoxycoumarin, whereas the highest solubility in SC is observedfor phenylethyl resorcinol . The solubilities of the different chemicals inSC at the same 97% RH  or the excesssolid one (WAXS). There are numerous other methods to study solubilityin SC presented in the literature. Many of these approaches use SCthat is fully hydrated or at an excess volume of a relatively diluteaqueous solution.38 The methodology presented here provides an important complementto these previous studies as it is applicable for a large range ofhydration conditions, also low RHs. In addition, it avoids the risksof extraction of SC components—in particular NMF and shorter-chainlipids19—to the solution that is in contact with theSC. Another advantage of the present method is that it is possibleto study the uptake of hydrophobic chemicals, good candidates forskin permeation,40 without the need to use organicsolvents that also influence the mobility of the SC lipid and proteincomponents.43 Finally, wepoint out that one can use the same experimental approach as usedhere for equilibrated samples for time-resolved studies of uptake of different chemicals into SC, as exemplified for2-MR in Figure S2.The present methodology makes it possibleto distinguish between the fraction of the added compound that isactually dissolved inside the SC and an excess fraction of the samecompound that is not taken up in the SC but may still be depositedon its surface or in furrows. This methodology is based on the detectionmethods that act as a dynamic filter to spot only one of the fractionseither the dissolved fraction in SC NMR experiments44 only show the 13C signals of the dissolved fraction of the added chemical with C-Hbond rotational correlation time faster than ca. 10 ns.45 One can also use the more time-consuming Q (quantitative)-INEPT method13 to quantify the amount of solubilized added chemical compared withthe amount of SC mobile components in a sample with an excess amountof added solid chemical. Another possible approach is fluorescencespectroscopy using a fluorophore of whose spectra can be shifted whenchanging its local environment.46 Finally,it is noted that the detection limit of these approaches can varybetween chemicals due to differences in the NMR and WAXS spectra ofthe pure chemicals in combination with SC (or delipidized SC) as wellas differences in instrumental setups with different measurement timesand/or beam intensities. We also note that both NMR and WAXS spectradiffer between SC and delipidized SC, meaning that the detection limitwhere signals from the added compound can be resolved may vary betweenthe different types of samples.One requirementfor the present methodology is that the pure chemical is present inits solid form at the investigated hydration and temperature. If thecompound is dissolved in the excess water that is outside SC, thiswould interfere with the measurement since it is then not possibleto distinguish between the chemical that is dissolved in SC and thatdissolved in any excess solution. For the latter cases, the solubilityin SC can still be estimated if one also knows the solubility of thechemical in the solvent as well as the total volume of the excesssolution. The added chemical should also be compatible with the detectionmethods. The use of 3.5PO/W of the chemicals together with their solubilityin the formulation of interest . Inted data 1, it is nditions 1. The sont in SC 110,12,14ted here 1. It is w = RT ln RH), the water chemicalpotential, Δμw, is the same in all parts ofthe SC sample (R is the gas constant and T is the temperature).The present methodologycan be extended to also determine the distribution of an added chemicalbetween different regions of a complex biomaterial by measuring thesolubility in each of the regions in conditions that correspond tothe same equilibrium. With respect to the present system, it is possibleto extract the distribution of an added compound between the corneocytesand the extracellular lipids within the SC. This requires separatemeasures of the solubility experiments for SC, delipidized SC, andextracellular lipids in conditions where the chemical potentials ofthe different components are the same for all experiments. As outlinedin 13 and the uptake of the chemical will naturallyvary between the co-existing solid and fluid lipid domains. In general,the solubility of any chemical is expected to be much higher in afluid matrix compared to a solid. The average solubility in the SClipids therefore will be much lower compared to the solubility influid lipids only, which would be more similar to the octanol solution.In addition, the SC lipids form self-assembled structures with apolarand polar domains within the lamellar arrangement, which cannot becaptured by a simple octanol solution. This will be relevant in particularto the dissolution of amphiphilic and polar molecules in the SC lipiddomains. Turning to the delipidized SC, the measured proportions ofthe added compounds and water inside these samples may exceed thesaturation concentration in a water solution . For caffeine, the solubility in porcine andhuman SC at full hydration was estimated from literature data55 to be 2.8–4.3 wt % (using D2O), which is higherbut still in the same range as the herein presented value of 2.4 wt% at RH 97% (using D2O, 10 The observeddifferences are thus in line with the previous reported data. It isalso noted that the reported values of partition coefficients wereobtained from the experiments where the chemicals were added togetherwith organic solvents,55 which may also influence theSC fluidity52 and thereby also the SC abilityto dissolve the added chemicals.Most of previous studies on solubility in SC report on the partitioncoefficients of added chemicals between SC and an excess aqueous solutions456 using Franz diffusioncell. In this case, the chemical is noted to be present at a saturatedconcentration or an activity of unity, corresponding to the highestdriving force for skin penetration. The same approach to measure solubilitycan be applied to other systems with low water content. Other applicationsmay be found in dry formulations in food, drug delivery, and cosmetics,where the solubility of constituents in the formulation changes duringfreeze-drying or exposure to dry air, resulting in segregation orprecipitation.Based on the here presentedmethodology, one can measure the solubilityin SC and delipidized SC, and one may also obtain estimate of thedistribution of the added chemical between the extracellular lipidsand the corneocytes at any hydration condition. The increase in solubilityin SC and the redistribution of the added chemicals between the differentlipid and corneocyte regions at varying hydration conditions can havea large impact on SC permeability. These variations can also impactthe dermal chemical safety and efficiency assessment if not performedat relevant hydration conditions. The experiments further demonstratethat rather large amounts of solid chemicals can actually be dissolvedin the SC on a minute timescale, pointing at the potential risks relatedto accidental exposure to such solid chemicals. This is also in linewith previous studies