Peter Matthew

Paul T Fowler

Gian Vincent Dizon

○ Lemmuel

Lemmuel L Tayo

Alvin R Caparanga

James Huang

Jie Zheng

Pierre Aimar

Yung Chang
email: ychang@cycu.edu.tw

Surface Zwitterionization of Expanded Poly(tetrafluoroethylene) via Dopamine-Assisted Consecutive Immersion Coating

Keywords: expanded polytetrafluoroethylene, dopamine, polyethylenimine, sulfobetaine methacrylate, glycidyl methacrylate

Expanded polytetrafluoroethylene (ePTFE) is one of the materials widely used in the biomedical field, yet its application is being limited by adverse reactions such as thrombosis when it comes in contact with blood. Thus, a simple and robust way to modify ePTFE to be biologically inert is sought after. Modification of ePTFE without high-energy pretreatment, such as immersion coating, has been of interest to researchers for its straightforward process and ease in scaling up. In this study, we utilized a two-step immersion coating to zwitterionize ePTFE membranes. The first coating consists of the codeposition of polyethylenimine (PEI) and polydopamine (PDA) to produce amine groups in the surface of the ePTFE for further functionalization. These amine groups from PEI will be coupled with the epoxide group of the zwitterionic copolymer, poly(GMA-co-SBMA) (PGS), via a ring-opening reaction in the second coating. The coated ePTFE membranes were physically and chemically characterized to ensure that each step of the coating is successful. The membranes were also tested for their thrombogenicity via quantification of the blood cells attached to it during contact with biological solutions. The coated membranes exhibited around 90% reduction in attachment with respect to the uncoated ePTFE for both Gram-positive and Gram-negative strains of bacteria (Staphylococcus aureus and Escherichia coli). The coating was also able to resist blood cell attachment from human whole blood by 81.57% and resist red blood cell attachment from red blood cell concentrate by 93.4%. These ePTFE membranes, which are coated by a simple immersion coating, show significant enhancement of the biocompatibility of the membranes, which shows promise for future use in biological devices.

■ INTRODUCTION

Polytetrafluoroethylene (PTFE) is an interesting fluoropolymer in that its chemical nature, particularly the strength of its carbon-fluorine bonds and the smooth protective electron sheath provided by the fluorine atoms, results in exceptional chemical inertness, high thermal stability, and wear resistance. [START_REF] Bunn | Structures of Molecules and Crystals of Fluoro-Carbons[END_REF] When subjected to anisotropic expansion, the resulting expanded PTFE (ePTFE) retains most of the desirable properties of bulk PTFE while introducing pores of controllable size, known to imbue the polymer with additional strength and flexibility as well as potential for tissue and blood vessel in-growth. [START_REF] Ebnesajjad | Characteristics, and Applications of Expanded PTFE (ePTFE) Products[END_REF] These characteristics have made ePTFE the most commonly used fluoropolymer in medical applications, particularly in large-diameter, high-flow vascular grafts, [START_REF] Faries | A comparative study of alternative conduits for lower extremity revascularization: Allautogenous conduit versus prosthetic grafts[END_REF][START_REF] Kannan | Current status of prosthetic bypass grafts: A review[END_REF] and hemodialysis access grafts. [START_REF] Li | Neointimal hyperplasia associated with synthetic hemodialysis grafts[END_REF] Though chemically inert, ePTFE is not biologically inert, and this has severely limited its applications. For instance, small-diameter grafts made of ePTFE have not been successful in vivo due to the high thrombogenicity of the material and poor endothelialization, [START_REF] Harris | Evaluation of Polytetrafluoroethylene Micrografts in Microvascular Surgery[END_REF][START_REF] Tseng | Effects of amide and amine plasmatreated ePTFE vascular grafts on endothelial cell lining in an artificial circulatory system[END_REF] resulting from excessive serum protein and platelet adhesion; [START_REF] Greisler | Interactions at the Blood/Material Interface[END_REF] this poses the limit of only using medium-to large-diameter ePTFE grafts to prevent occlusion by thrombi and subsequent complications. Bacterial infections have also been a pressing issue in ePTFE grafts and surgical meshes, [START_REF] Nassar | Infectious complications of the hemodialysis access[END_REF][START_REF] Benrashid | Operative and perioperative management of infected arteriovenous grafts[END_REF][START_REF] Kirkton | Susceptibility of ePTFE vascular grafts and bioengineered human acellular vessels to infection[END_REF] with Gram-positive Staphylococcus aureus being responsible for majority of these cases. [START_REF] Bachleda | Infected Prosthetic Dialysis Arteriovenous Grafts: A Single Dialysis Center Study[END_REF] This has been attributed to both the hydrophobicity of the surface and the accessibility of the micropores in the material. [START_REF] Melman | Histologic and biomechanical evaluation of a novel macroporous polytetrafluoroethylene knit mesh compared to lightweight and heavyweight polypropylene mesh in a porcine model of ventral incisional hernia repair[END_REF] Most recent attempts at improving the biocompatibility of ePTFE are generally divided into two approaches: seeding endothelial cells in vitro or encouraging their in-growth in vivo, [START_REF] Lu | Antithrombotic and fibrinolytic system of human endothelial cells seeded on PTFE: the effects of surface modification of PTFE by ammonia plasma treatment and ECM protein coatings[END_REF][START_REF] Begovac | Improvements in GORE-TEX vascular graft performance by Carmeda bioactive surface heparin immobilization[END_REF][START_REF] Tzchori | Improved Patency of ePTFE Grafts as a Hemodialysis Access Site by Seeding Autologous Endothelial Cells Expressing Fibulin-5 and VEGF[END_REF][START_REF] Lei | Autologous Vascularization: A Method to Enhance the Antibacterial Adhesion Properties of ePTFE[END_REF] or surface modification by grafting molecules that confer biocompatibility. [START_REF] Zhu | Blood compatibility of chitosan/ heparin complex surface modified ePTFE vascular graft[END_REF][START_REF] Larsen | A biomimetic peptide fluorosurfactant polymer for endothelialization of ePTFE with limited platelet adhesion[END_REF][START_REF] Sisti | Antibacterial coatings on poly-(fluoroethylenepropylene) films via grafting of 3-hexadecyl-1-vinylimidazolium bromide[END_REF][START_REF] Venault | Biofouling-resistance control of expanded poly(tetrafluoroethylene) membrane via atmospheric plasma-induced surface PEGylation[END_REF][START_REF] Liu | A surface graft polymerization process on chemically stable medical ePTFE for suppressing platelet adhesion and activation[END_REF][START_REF] Liu | Anti-platelet adhesion and in situ capture of circulating endothelial progenitor cells on ePTFE surface modified with poly(2-methacryloyloxyethyl phosphorylcholine) (PMPC) and hemocompatible peptide 1 (HCP-1)[END_REF] Surface modification is also used to encourage endothelialization. [START_REF] Zheng | Mussel-inspired triblock functional protein coating with endothelial cell selectivity for endothelialization[END_REF] To modify the surface of ePTFE membranes, processes that are energetic enough to lyse the strong C-F bonds or introduce reactive groups have been used extensively. These include plasma treatment, atomic layer deposition, and irradiation by ions, γ rays, or UV light. All of these have the common features of complexity, expensive and delicate instrumentation, and high energy input. [START_REF] Feng | Progress and perspectives in PTFE membrane: Preparation, modification, and applications[END_REF][START_REF] Cheng | Surface functionalization of polytetrafluoroethylene substrate with hybrid processes comprising plasma treatment and chemical reactions[END_REF] Wet-chemical modification, where the substrates are simply immersed in a reactive solution, has gained much attention recently primarily due to the need for more facile processes of surface modification, despite coming with the disadvantage of producing potentially toxic waste solutions; in the light of energy consumption and process simplicity, it is however a desired option. Previous approaches have used strong solutions such as oxidizing mixtures of nitric acid and potassium permanganate that introduced more reactive hydroxyl (C-OH) and carbonyl (CO) groups. [START_REF] Wang | Surface modification of porous poly(tetrafluoraethylene) film by a simple chemical oxidation treatment[END_REF][START_REF] Fu | Investigation on surface structure of potassium permanganate/nitric acid treated poly-(tetrafluoroethylene)[END_REF] More recently, biomimetic mussel-inspired approaches based on dopamine self-polymerization and adhesion have been successful at introducing various molecules onto the otherwise nonstick ePTFE surface. [START_REF] Xi | A facile method of surface modification for hydrophobic polymer membranes based on the adhesive behavior of poly(DOPA) and poly(dopamine)[END_REF] Progress since this discovery consists of altering the solvent for the coating solution, [START_REF] Li | Mussel-inspired modification of PTFE membranes in a miscible THF-Tris buffer mixture for oil-inwater emulsions separation[END_REF] co-deposition or conjugation of other molecules with dopamine, dopamine coating followed by grafting of a second coating via reactive groups in polydopamine, [START_REF] Ma | Anchorable phosphorylcholine copolymer synthesis and cell membrane mimetic antifouling coating fabrication for blood compatible applications[END_REF] or the combination of the last two methods. For example, dopamine-hyaluronic acid conjugates were successfully coated onto PTFE under mild conditions at room temperature and mildly basic pH, increasing its hydrophilicity and resistance to protein and macrophage cell adhesion and scarring in mouse tissue in vivo. [START_REF] Lee | Universal surface modification using dopamine-hyaluronic acid conjugates for antibiofouling[END_REF] Polyethyleneimine (PEI) has also been co-deposited with dopamine to form a polydopamine/PEI (PDA/PEI) coating on porous ePTFE for use in microfiltration. This also improved the hydrophilicity and water flux through the membrane. Moreover, the coating was found to be stable in a wide range of pH values. [START_REF] Song | Durable hydrophilic surface modification for PTFE hollow fiber membranes[END_REF] In a related study, a structurally similar compound, catechol, was used instead of dopamine. This was conjugated to PEI, then coated onto PTFE. A second step introduced a silane group by covalent linkage between the first layer and the reactive epoxy group of the silane-containing compound. This endowed the membrane with superior hydrophilicity, allowing higher water fluxes in their oil-water separation experiments. [START_REF] Xue | A catechol-based biomimetic strategy combined with surface mineralization to enhance hydrophilicity and anti-fouling property of PTFE flat membrane[END_REF] A similar approach used gallic acid (3,4,5-trihydroxybenzoic acid) instead, conjugated to PEI with subsequent covalent attachment of a silane moiety via the nucleophilic opening of an epoxy group. In addition to increased hydrophilicity, the coating was also effective against Escherichia coli and S. aureus. [START_REF] Li | One-bath two step method combined surface micro/nanostructures treatment to enhance antifouling and antibacterial property of PTFE flat membrane[END_REF] Despite the multitude of studies on this fast and robust method of modifying ePTFE surfaces for increased hydrophilicity and antibacterial properties, no such effort has been made in studying the hemocompatibility of ePTFE membranes modified this way.

We present in this study a facile and low-energy two-step surface zwitterionization procedure using a mussel-inspired dopamine dip-coating approach that could successfully convert the hydrophobic ePTFE surface into a highly hydrophilic one with enhanced biocompatibility and that could be potentially used to improve the performance of ePTFE in biomedical applications. We tested different PEI molecular weights to determine an optimum coating procedure for the greatest improvement in biocompatibility. The modified membranes were tested for their hydrophilic character, surface morphology, surface chemistry, and resistance to fouling by bacterial and blood cell solutions in attachment tests. We have also attempted to provide meaningful correlations that could be referred to in future designs for hemocompatible biomaterials. To the best of our knowledge, this is a pioneering study on using a low-energy mussel-inspired coating procedure on a highly hydrophobic material to achieve both antibacterial property and hemocompatibility by surface zwitterionization.

■ EXPERIMENTAL SECTION

Materials and Reagents. Chemicals such as branched PEI (0.6 and 10 kDa), dopamine hydrochloride, Tris base, and 2,2′-azobis-(2methylpropionitrile) (AIBN) were purchased from Sigma-Aldrich; branched PEI (1.8 kDa) was purchased from Alfa Aesar; glycidyl methacrylate (GMA) was purchased from Tokyo Chemical Industry Co., Ltd.; 25 wt % aqueous solution of glutaraldehyde was purchased from Acros Organics and was diluted to 2.5 wt % with deionized water before use; and 1 M hydrochloric acid (HCl) was bought from Honeywell Fluka. Sulfobetaine methacrylate (SBMA) monomer was synthesized in accordance with Yue et al. [START_REF] Yue | Grafting of zwitterion from polysulfone membrane via surfaceinitiated ATRP with enhanced antifouling property and biocompatibility[END_REF] The ePTFE membranes were purchased from EF-Materials Industries, Inc. HPLC-grade solvents such as methanol, n-butanol, and isopropanol (IPA) were purchased from Aencore Chemical Pty., Ltd. and Echo Chemical Co., Ltd. Phosphate-buffered saline (PBS) was first diluted to 1x from 10x concentration before use.

Preparation of ePTFE Membranes. The ePTFE membranes were cut into 0.85 cm disks and immersed in IPA to wash the membranes from dust and other dirt. The washing process was performed by putting the membranes under sonication three times for 15 min each time with replacement of the isopropanol every time. The membranes were then stored at 4 °C in IPA until further use.

Synthesis of Poly(GMA-co-SBMA). Poly(GMA-co-SBMA), which will be denoted as PGS, was synthesized according to the protocol of Chou et al., [START_REF] Chou | Zwitterionic surface grafting of epoxylated sulfobetaine copolymers for the development of stealth biomaterial interfaces[END_REF] where an optimal GMA/SBMA ratio of 20:80 was used. Briefly, predetermined amounts of the monomers were dissolved: SBMA in deionized water, and GMA in methanol. These two solutions were then mixed, followed by addition of AIBN as a free-radical initiator. The solution was stirred for at least 20 min to ensure that the solution is completely homogeneous. The reaction solution was then placed in an oil bath controlled at 60 °C, and the reaction was kept running for 6 h. Polymerization was thermally terminated by transfer of the reaction flask to an ice bath. The copolymer was purified with methanol at least three times via reprecipitation. It was then dried under vacuum overnight to remove the bound solvent. Finally, freeze drying and grinding yielded the white crystalline PGS powder.

Coating with Dopamine and PEI. The overall coating process is shown in Scheme 1. Tris buffer was initially prepared by dissolving Tris base in deionized water to a final concentration of 50 mM and then adjusting the pH of the solution to 8.5 using HCl. Dopamine and branched PEI were dissolved in the Tris buffer with concentrations of 2 and 1 mg/mL, respectively. This dopamine/PEI mass ratio has been adopted from Lv et al., whose study has shown that it was the optimum ratio based on the thickness of the coating produced. [START_REF] Lv | Co-deposition Kinetics of Polydopamine/Polyethyleneimine Coatings: Effects of Solution Composition and Substrate Surface[END_REF] Upon dissolution, the dopamine immediately started undergoing selfpolymerization into polydopamine (PDA), reacting also with PEI to form PDA/PEI conjugates. The ePTFE disks were immersed in a freshly prepared PDA/PEI coating solution in 2 mL Eppendorf tubes. The tubes were kept in a 37 °C oven for 24 h. Subsequently, the membranes were removed from the solution and washed three times with deionized water. Three different PEI molecular weights were used in this study: 0.6, 1.8, and 10 kDa.

Zwitterionization of ePTFE with PGS Copolymer. The coating solution for the zwitterionization process was prepared by dissolving the PGS copolymer in deionized water preheated to 60 °C, to a concentration of 10 mg/mL. The PDA/PEI-coated ePTFE disks were immediately immersed in the PGS coating solution immediately after washing with deionized water. The zwitterionization of the ePTFE disks was done and kept at 60 °C for 24 h. Membranes coated with only the PDA/PEI coating solution were labeled as D-P600, D-P1800, and D-P10000; membranes coated with both PDA/PEI and PGS coating solutions were labeled as D-P600-GS, D-P1800-GS, and D-P10000-GS. The coated samples were then stored immersed in deionized (DI) water until further use.

Physical Characterization of Coated Membranes. Coating density (mg/cm 2 ) was measured as the difference in dry mass before and after coating, normalized to the external surface area of each membrane. Water contact angle measurements were done with a DataPhysics OCA15EC goniometer to assess the surface hydrophilicity of the virgin and coated membranes. A 4 μL droplet was slowly added to the surface of each membrane. A dynamic measurement of the water contact angle was performed, keeping track of the change in contact angle while the droplet is being absorbed by the membrane. Measurements were done in triplicate for each condition. Differences in the slopes of the curves directly relate to hydrophilicity of the coatings. In conjunction with this, hydration tests were also performed to assess the hydrophilicity of the internal microstructure of the modified membranes. Preweighed samples were immersed in 1 mL of deionized water and stored for 24 h. The membranes were then gently pat-dried with laboratory-grade clean wipes to remove water adhered to the surface and weighed immediately; the difference in mass was assumed to be the mass of water absorbed by the membrane. Normalizing the water absorbed by the membrane to the membrane surface area gave hydration capacity (mg/cm 2 ). A porosity test was performed to determine if the overall procedure has affected the porosity of ePTFE, following a similar procedure except that the membranes were immersed in 1 mL of nbutanol. The alcohol was assumed to fill up the pores within the membrane. The following formula was used to estimate the porosity of the membranes

ρ ρ ρ ρ = - - - m m m m porosity (%) ( ) ( ) M W D M W S M D
where ρ M is the bulk density of the membrane (PTFE, 2.2 mg/cm 3 ), ρ S is the density of the solvent used (n-butanol, 0.81 mg/cm 3 ), and m W and m D are the wet and dry masses of the membranes, respectively. In both the hydration and porosity tests, triplicate measurements were performed, and the data obtained were averaged. Changes in the surface and internal microstructure were investigated by visualizing the surface and cross sections, respectively, using a scanning electron microscope (SEM). The samples were sputtered with gold and then visualized with a Hitachi S-3000 SEM at an accelerating voltage of 3 keV. Surface Chemical Characterization of Coated Membranes. Coated membrane samples were subjected to X-ray photoelectron spectroscopy (XPS) analysis to confirm the presence of the PDA/PEI and PGS layers on the surface of the membranes. XPS analysis was performed using a Thermo Scientific K-Alpha X-ray photoelectron spectrometer System. A narrow scan in the C 1s, S 2p, and N 1s regions was done to confirm sulfur and nitrogen coming from the sulfobetaine functional group of PGS.

Biocompatibility Tests for the Coated Membranes. Fouling resistance tests were performed to evaluate biocompatibility. Membranes were exposed to biological fluids such as human whole blood and the erythrocyte-rich fraction, and their extent of attachment was investigated. Human whole blood collected from a pool of healthy individuals was provided by MacKay Memorial Hospital (Taipei, Taiwan). Virgin and coated membrane samples were individually placed in the wells of a sterile 24-well plate, immersed in PBS, and incubated at 37 °C for at least 4 h before each attachment test. The biological fluid samples were also kept at the same temperature just prior to use. After removal of the PBS in each sample, each membrane was immersed in 1 mL of the respective biological fluid for 1 h at 37 °C. The membranes were then washed with fresh PBS and fixed with 1 mL of a 2.5 wt % glutaraldehyde solution overnight.

Bacterial attachment tests were also performed to assess resistance to fouling by microorganisms forming biofilms on the samples. Grampositive S. aureus and Gram-negative ATCC 23225 E. coli were used. E. coli cells were cultured by inoculating 1.8 mL of a preserved bacterial solution having a concentration of 8 × 10 8 cells/mL into 50 mL of sterile medium made up of 3 g/L beef extract and 5 g/L Bacto Peptone for 12 h. S. aureus cells were cultured in a 25 g/L Lysogeny broth (LB) medium and grown for 15 h. The solutions were both incubated at 37 °C with constant shaking at 100 rpm. Prior to the attachment tests, membranes were prepared in the same way as with the blood attachment tests. Briefly, the samples were incubated in PBS at 37 °C for at least 4 h prior to each test. The membranes were then immersed in 1 mL of each bacterial solution and incubated for 24 h at the same settings, with replacement of fresh bacterial solutions every 12 h. After the attachment experiment, the samples were washed with PBS three times, and then 1 mL of 2.5% glutaraldehyde was introduced in each sample to fix the attached cells. For all of the biocompatibility tests, the glutaraldehyde solution induces fluorescence of the fixed cells, which renders them visible under a confocal laser scanning microscope (CLSM A1R+, Nikon), using excitation and emission wavelengths of 488 and 520 nm, respectively. Images were analyzed with ImageJ software, which allowed quantification of the relative area covered or occupied by the cells. Two images were captured for each membrane, and there were three replicates for each coating condition; thus, a total of six images were obtained and averaged for each condition. Virgin ePTFE membranes and SBMA hydrogels were used as negative and positive controls, respectively, in the biocompatibility tests.

■ RESULTS AND DISCUSSION

Surface Physical Characterization of Samples. The chemical inertness of PTFE is a direct consequence of the high bond dissociation energy of the C-F bonds, in addition to the protective electron sheath of the fluorine atoms covering the internal carbon backbone of the polymer molecules. This property makes it difficult to modify ePTFE using the common grafting procedures done for other polymeric surfaces. Surface modification using the strong adhesive properties of PDA https://dx.doi.org/10.1021/acsami.0c09073 bypasses the need for a direct covalent linkage onto ePTFE, as it coats the polymer via a combination of hydrogen-bonding and pi-electron interactions. Because the procedure relies on the success of the first coating step, multiple chemical tests must be performed to confirm the presence of these coating layers.

After the first coating with PDA/PEI, there was a negligible change in the membranes' weights, as shown in Figure 1.

There were, however, notable changes in membrane color: from white for virgin ePTFE to brown for the coated samples seen in the images of the membranes in Figure 1. The darkest color was seen for D-P600, and with higher molecular weights of PEI, the color became less intense. It is worth noting that only PDA contains a chromophore and PEI does not; the variation in membrane color is therefore directly indicative of the amount of PDA adhering to the surface as a result of interacting with PEI. [START_REF] Yang | Mussel-inspired modification of a polymer membrane for ultrahigh water permeability and oil-in-water emulsion separation[END_REF] Additionally, since all of the coating solutions were also brown and transparent at the end of the coating, some undeposited PDA/PEI conjugates remain in solution. These direct observations point to the effect of the molecular weight of PEI used, where lower molecular weights were seen to be better at enhancing the deposition of PDA/ PEI conjugates onto the substrate surface. Although this same trend has been observed in previous studies involving PDA/ PEI co-deposition on different surfaces, [START_REF] Lv | Co-deposition Kinetics of Polydopamine/Polyethyleneimine Coatings: Effects of Solution Composition and Substrate Surface[END_REF][START_REF] Yang | Effects of polyethyleneimine molecular weight and proportion on the membrane hydrophilization by codepositing with dopamine[END_REF] different explanations have been used to justify the effect of PEI molecular weight on deposition. This is indeed an interesting outcome, considering that despite the differences in molecular weight, the usage of the same mass of PEI in the experiments should mean equivalent amounts of the reactive amine groups in each case, and consequently, a consistent reaction stoichiometry between the PDA and these amine groups. It must be noted, however, that with the longer chains of higher-molecular-weight PEI, there is an increasing degree of both intermolecular and intramolecular interactions, aside from the larger molecules having a much lower diffusivity in solution. Thus, despite following the same Schiff base or Michael addition stoichiometry as that in lower-MW PEI, the longer chains of higher-MW PEI in the formed PDA/PEI conjugates encourage more interactions within the same molecule instead of with the substrate surface. This in effect "buries" the adhesive groups of PDA within the conjugates, causing stabilization in the aqueous phase and resulting in an overall decrease in the amount of PDA/PEI conjugates deposited. [START_REF] Lv | Co-deposition Kinetics of Polydopamine/Polyethyleneimine Coatings: Effects of Solution Composition and Substrate Surface[END_REF] This is reinforced by observations from succeeding physicochemical characterizations.

After the second coating, which formed the next layer consisting of PGS covalently linked to the first layer, the coating density was seen to increase drastically. Relative to the first layer, this second layer of PGS was thicker and more massive by several micrograms.

The SEM images shown in Figure 2 reveal the effect of coating on the surface morphology of the ePTFE; the crosssectional views of the membranes are shown as inset images. Virgin expanded PTFE is characterized by nodes and fibrils as a result of the expansion process. Despite not having seen any change in weight after the PDA/PEI coating, SEM images reveal that there is an observable slight change in the surface morphology. The pores of the virgin ePTFE were slightly covered after the PDA/PEI coating but were virtually unchanged after the zwitterionization with PGS. More surface morphological changes are visible for the low-MW PEI after only the first coating, compared to the much larger 10 kDa PEI. This is consistent with the earlier explanation that there is less deposition of PDA/PEI conjugates with high-MW PEI. Further confirmation comes from the morphological changes after the second coating, where the greatest change is observed with D-P600-GS and the least with D-P10000-GS. This change obviously comes from the attachment of GS. With much less PDA/PEI deposited onto the surface of the D-P10000, less GS could attach; conversely, with the higher amount of PDA/PEI on the surface of D-P600, there are more sites for the attachment of GS. These SEM images, particularly those for the GS-coated samples, seem to suggest pore blockage at the surface, which could potentially alter the bulk physical properties of the membrane. To test whether these morphological changes are significant to the overall microstructure of the membrane, porosity tests based on swelling by n-butanol were performed. Interestingly, the porosities were not found to be significantly different from the porosity of the virgin membrane, as presented in Table 1. In fact, the crosssectional SEM images show that aside from the deformities introduced by the tearing of the membranes to reveal the interior, the nodes are fibrils characteristic of ePTFE are still visible. For D-P1800-GS, these are visible at the top-right corner. The large "flat" region may have resulted from the tearing; porosity data for this membrane are not consistent with blockage. Good coats must only alter surface properties and not bulk properties; in this case, porosity, a representative bulk property, was not altered, and hence in this regard, the coats are not problematic.

Surface Chemistry of Modified Membranes. In the first coating, dopamine self-polymerizes and randomly reacts with amine moieties in PEI to form a co-deposited layer of PDA/ PEI on the membrane surface. [START_REF] Lv | Co-deposition Kinetics of Polydopamine/Polyethyleneimine Coatings: Effects of Solution Composition and Substrate Surface[END_REF] As mentioned previously, the stoichiometry of this reaction, in terms of moles of reactive amine groups, is unaffected by PEI molecular weight. In the second coating, the epoxy rings of the PGS are attacked and opened by the unreacted nucleophilic amine groups in PEI (mostly secondary and tertiary amines not participating in the Schiff base and Michael addition reactions earlier), forming a covalently linked second layer. The first layer, composed of codeposited PDA and PEI, serves two functions: to form a tight noncovalent surface coating via the free catechol groups inherent in the polydopamine structure, and to introduce the nucleophilic amino group for the subsequent attachment of the PGS copolymer via a ring-opening nucleophilic attack on the glycidyl moieties shown in Scheme 2. This second layer presents free sulfobetaine groups, which are zwitterionic moieties. The PDA/PEI layer in itself has contributed polar functional groups (CO, C-OH, CN, R 3 -N, R 2 -NH) that could interact with water, but the full charges present in PGS render the layer even more hydrophilic by stronger Coulombic interactions with polar water molecules. It is thus reasonable to hypothesize that with more PGS on the surface, the more hydrophilic the surface should become, the stronger the hydration layer is held.

The success of the coating was also confirmed by XPS measurements on the virgin membrane and the samples D-P600 and D-P600-GS. Figure 3 shows the C 1s, N 1s, and S 2p spectral scans. The C 1s spectrum for virgin PTFE shows a strong -CF 2 peak (291.7 eV). Addition of the PDA/PEI layer significantly enhanced the C-C band at 284 eV and introduced two new bands at 285.6 eV (C-O and C-N) and 288 eV (CO) as well as a peak in the N 1s spectrum at 398.9 eV. [START_REF] Song | Durable hydrophilic surface modification for PTFE hollow fiber membranes[END_REF] The new carbon peaks come from the polydopamine structure (Scheme 2), which contains oxygen both singly and doubly bonded to carbon in the catechol groups, and the nitrogen peak comes from the amine groups of both PDA and PEI. Finally, the second PGS layer further introduced a sulfur peak at 166.6 eV (-SO 3 -) and a new ammonium nitrogen (R 4 -N + ) peak at 401.4 eV, which could only come from its sulfobetaine group and which confirms successful PGS grafting. In addition, relative to the PDA/PEI coating, the PGS coating has significantly decreased the intensity of the -CF 2 peak as more C-C (mostly alkyl) carbons have been added. A summary of the elemental analysis is given in Table 2.

Evaluation of the Antifouling Properties of the Modified ePTFE Membranes. Dynamic water contact angle measurements, summarized in Figure 4a as a plot of contact angle versus time, show that virgin ePTFE is strongly hydrophobic, having a contact angle of 130°. The curves for D-P600-GS and D-P1800-GS are shown as highly hydrophilic as the water droplet was very quickly absorbed by the membranes, showing only a few contact angles before reaching a value of zero. Without PGS, hydrophilicity was strongly dependent on the PEI molecular weight used: D-P600 was seen to be the most hydrophilic based on the low contact angle, followed by D-P1800, then by D-P10000. This is again consistent with the hypothesis that high-MW PEI, despite possessing the same amount of reactive amine groups, interferes with the deposition process by promoting intramolecular interactions that stabilize the conjugates in solution instead of encouraging their deposition onto the surfaces. As a result, there are more PDA/PEI conjugates deposited on D-P600, slightly less on D-P1800, and so much less on D-P10000. In fact, both the value and the behavior of the contact angle for D-P10000 were close to those of the virgin PTFE, pointing to poor deposition on the surface. Additionally, the water droplet was most quickly absorbed through D-P600 and least through D-P10000. This means the pores in the interior of the membrane were more successfully coated with lower MW of PEI. This is confirmed by the hydration capacities (Figure 4b): more water is bound by the more hydrophilic pores of D-P600 and decreases with increasing PEI molecular weight. Addition of the PGS layer further improved the hydrophilicity, but for D-P10000-GS, this improvement was not as significant. The almost similar hydration capacities for all PGS-coated membranes point at saturation effects such that the differences in PDA/PEI density forming the first layer were normalized by the saturation of zwitterion-water interactions at the surface after the second coating. PGS is itself already a polymer, and as such, it requires only few covalent linkages per polymeric unit for it to remain attached to the first layer. The saturation effect is explained best when considering that as long as the PGS has been attached to the surface with at least a few covalent linkages, there would be no significant difference with a PGS unit being attached with more linkagesthe membrane has already been rendered zwitterionic. For example, despite being less hydrophilic than D-P600, D-P1800 has been rendered as hydrophilic after GS coating such that both D-P600-GS and D-P1800-GS have the same hydration capacity. The case for D-P1000 warrants more discussion. It must be noted that the time scales for both experiments differseconds for the contact angle test and a whole day for the hydration capacity. The dynamic water contact angle for D-P10000-GS has a nonzero slope, and it decreases very slowly, unlike that for D-P10000enough evidence that some GS has successfully attached. Besides, the coating density for D-P10000-GS was nonzero (Figure 1), although it must be emphasized that these values are nevertheless very small, being in micrograms. With this nonzero slope, D-P10000-GS can still absorb as much water as it can overnight until it is saturated. In summary, the whole coating procedure successfully enhanced the hydrophilicity of the PTFE membranes, to only slightly different extents. The hydrophilicity of the material is a good indication of its antifouling property. The biofoulant would need to displace the tightly bound water molecules on the zwitterionic polymer chains. Thus, the samples were further subjected to experiments to evaluate their antifouling ability.

To assess how the modification affects the performance of the membrane against supporting biofilm formation, which would cause unwanted and sometimes fatal bacterial infections, bacterial attachment tests were performed. It has been hypothesized that biofilm formation is a stepwise process, beginning with the most important step of forming a conditioning film, which is composed of organic and inorganic compounds that immediately adhere to surfaces exposed to bacteria-contaminated solutions. [START_REF] Matin | Biofouling in reverse osmosis membranes for seawater desalination: Phenomena and prevention[END_REF] This fouling was also seen to be more dominant on less hydrophilic surfaces, [START_REF] Fane | A review of fouling and fouling control in ultrafiltration[END_REF] strongly suggesting that fouling could be reduced with more hydrophilic surfaces. The antifouling activity of the modified membranes was observed with both E. coli and S. aureus, as shown in Figure 5, representing the Gram-negative and Grampositive groups of bacterial cells. Without any modification, virgin PTFE is prone to fouling by the microorganisms due to its hydrophobicity. In contrast, the SBMA hydrogel, which acted as the positive control for all biofouling experiments, shows essentially no attachment of either microorganism, owing to its high hydrophilicity forming a protective hydration layer. [START_REF] Holmlin | Zwitterionic SAMs that Resist Nonspecific Adsorption of Protein from Aqueous Buffer[END_REF] Results for both E. coli and S. aureus attachment for PDA/PEI-coated samples are consistent with the hydrophilic properties of the membranes. In fact, there is unquestionable consistency with the data from hydration capacity: D-P10000, being strongly hydrophobic and resisting water absorption, was the most fouled of the PDA/PEI-coated samples. Furthermore, it was also observed that after the PGS coating, the bacterial attachment was reduced significantly. With the PGS coating, a similar saturation effect is observed, as was the case for the hydration capacity of the modified membranes. The antifouling property provided by D-P10000GS seems to be in contradiction when considering only the water contact angle. Prior to exposing the membranes to the bacterial solutions, they were incubated in PBS at 37 °C for at least 4 h. As observed with the contact angle curve, the nonzero slope meant it could absorb water slowly. It is possible that by the time the D-P10000GS membranes were incubated with the bacterial solutions, they had already been saturated with water to provide the protective hydrating layer. The results so far confirm the direct consequence of hydrophilic character to antifouling tendencies, as it is known that hydrophilic surfaces more effectively prevent biofouling by promoting the formation of a protective hydrating layer. Furthermore, these results show that the coating is effective against both Gram-positive and Gramnegative microorganisms.

Successful prevention of bacterial attachment suggests the potential to prevent the attachment of other biological molecules. As ePTFE has consistently been used as materials for various grafts, which are constantly in contact with blood, the interaction of the modified membranes with blood was also studied. Such hemocompatibility studies are required for determining the biocompatibility of biomaterials designed for blood-interacting applications. To test this, attachment tests with whole blood and red blood cells (RBC)-rich samples were performed, and the results are shown in Figure 6. There is a stark difference in the fouling of membranes by RBCs and whole blood. For RBC attachment, the surface modification reduced the attachment of the red blood cells relative to virgin ePTFE; additionally, the GS-coated membranes performed better compared to those coated only with PDA/PEI. The trends in the antifouling performance for both PDA/PEI and GS-coated membranes, however, were the opposite of those seen in hydrophilicity. Fouling with whole blood is remarkable. Although a trend consistent with hydrophilicity is seen in the PDA/PEI-coated and in the GS-coated membranes, some of them were worse than virgin ePTFE at preventing fouling, even enhancing fouling 5-fold.

Ideally, the resistance of surfaces to biofouling is enhanced when surface hydrophilicity is increased. [START_REF] Rabe | Understanding protein adsorption phenomena at solid surfaces[END_REF] This has been attributed to the prevention of stochastic protein adsorption, which is believed to be the priming step in surface fouling. In whole blood, fibrinogen, a plasma protein, makes up the majority of the quickly absorbed plasma proteins; this is followed by attachment of platelets, inducing thrombogenesis. [START_REF] Wu | The role of adsorbed fibrinogen in platelet adhesion to polyurethane surfaces: A comparison of surface hydrophobicity, protein adsorption, monoclonal antibody binding, and platelet adhesion[END_REF] Since RBC concentrate is prepared from whole blood by centrifugation, which separates the much heavier cells from the much lighter plasma proteins, less to no fibrinogen is present in this RBC-rich fraction. Although membrane proteins do abound on RBCs, none of them are directly involved with the clotting process; [START_REF] Litvinov | Role of red blood cells in haemostasis and thrombosis[END_REF] instead, they are mostly involved in substrate transport and structural roles. However, it has been proposed that a group of these membrane proteins, called integrin-binding proteins because they bind to transmembrane integrin proteins responsible for cell adhesion to the extracellular matrix, presents a site for interaction with fibrinogen, [START_REF] Lominadze | Involvement of fibrinogen specific binding in erythrocyte aggregation[END_REF] suggesting that RBC attachment on surfaces is mediated by fibrinogen. Without much fibrinogen in this RBCrich fraction, the fouling observed could be speculated to be less dependent on the priming ePTFE-fibrinogen interactions, or that these interactions are too few and weak such that a secondary factor affecting biocompatibility has taken control. Following this logic, the excessive biofouling with whole blood could then be partly accounted for due to the abundance of fibrinogen.

This secondary factor affecting biocompatibility could be surface roughness, which is directly related to the presence of more surface irregularities and the total surface area exposed to the foulant. [START_REF] Vrijenhoek | Influence of membrane surface properties on initial rate of colloidal fouling of reverse osmosis and nanofiltration membranes[END_REF] Indeed, it has been shown that thicker PDA/PEI coatings are rougher as measured by atomic force microscopy. [START_REF] Lv | Co-deposition Kinetics of Polydopamine/Polyethyleneimine Coatings: Effects of Solution Composition and Substrate Surface[END_REF] This has been attributed to the accumulation of surface defects as layers grow thicker. Thus, with more deposition seen for PDA and 600 Da PEI, the formed coating is rougher; with 1800 Da PEI, it is less rough, while for 10 000 Da PEI, having the least amount of deposition, the surface is smoother. It is thus reasonable to attribute the RBC fouling seen for the PDA/PEI-coated membranes primarily to surface roughness due to the very low concentration of fibrinogen. The cells could more easily be stuck on rougher surfaces than on smoother surfaces. We say fibrinogen is present at a very low concentration instead of zero because it can be seen that the attachment of GS has still improved their fouling resistance. The zwitterionic moieties introduced could be thought of as preventing any surface attachment of the very few fibrinogen remaining, in addition to destabilizing weak RBC-ePTFE interactions.

Whole blood presents a harsher environment for the membranes, with normal concentrations of plasma proteins, particularly fibrinogen at 0.2-0.45 g/dL. Surface hydrophilicity becomes significant due to their effect on the formation of the priming protein layer. The trend in resistance indeed compares well with hydrophilicity: coats formed with 600 Da PEI provided better fouling resistance due to higher hydrophilicity, and those formed from 10 000 Da PEI were the most fouled. The saturation effect seen in hydration capacity, invoked to account for the resistance of all GS-coated membranes toward bacterial attachment, no longer holds for blood. It is possible that the osmolarity of whole blood due to its protein content causes the water bound on and in the membranes to diffuse out and into the blood. Thus, a hydration layer that is very loosely held could easily be destroyed when in contact with blood, whereas a strongly held layer can oppose this osmotic force. This could explain why D-P10000-GS did not fare wellit could not hold water as strongly as D-P1800GS and D-P600GS. This is also exactly why it absorbs water so slowly, as pointed out by the contact angle and hydration capacity tests (Figure 4).

The higher fouling of D-P1800, D-P10000, and D-P10000GS by whole blood relative to unmodified, virgin ePTFE could not be accounted for by hydrophilicity arguments; virgin ePTFE is more hydrophobic, but it was fouled less. It could not also be explained by surface roughness arguments, because then, D-P600, having the roughest surface, should have been fouled more than virgin ePTFE. Instead, it has resisted fouling better. The difference could be accounted for by considering the incubation step prior to exposure to the blood solutions. Being strongly hydrophobic, the PBS solution used for the incubation has been prevented from entering and wetting the interior pores of virgin ePTFE, whereas the other membranes, owing to their different hydrophilicities, allowed wetting of their interiors by the same solution. Blood can more easily penetrate pores that have already been pre-wet by the PBS than those that have not. The higher fouling for D-P1800, D-P10000, and D-P10000GS could then be because of the greater total surface area of these membranes in contact with blood, including its pores. This reconciles well with the hydrophilicity trend as seen for the GS-coated samples. All of these membranes could be saturated with the PBS solution, but D-P600-GS has even its interior pores coated well with PDA/PEI and GS, whereas D-P10000-GS has not. Finally, it could also be assumed that the amount of water that can be absorbed by D-P10000 (Figure 4) is so low that it falls below the milligram range, but is nonzero to still allow partial wetting of its pores by PBS. With this assumption in place, the observations with whole blood are now accounted for.

Despite the complexity of the interactions seen in the blood tests, both hydrophilicity and biocompatibility tests point at D-P600-GS as the best modified sample in this study. The low molecular weight of PEI ensures more PDA/PEI conjugates are deposited onto the ePTFE surface, which then provide a strongly adhering coating. Although biocompatibility largely relies on hydrophilicity for the protective hydration layer, it was shown in this study that surface morphology also becomes a significant factor in determining biocompatibility in the absence of a large amount of fouling proteins.

It is interesting to note the differences in fouling between the bacterial solutions and whole blood. Both attachment processes in these cases are dependent on the formation of a priming or activating layer, which is mostly proteinaceous. It can be hypothesized that compared to the normal physiological concentration of fibrinogen in blood (0.2-0.45 g/dL), proteins are less abundant in the bacterial solutions prepared and used, explaining why even loosely held hydration layer assumed for D-P10000-GS was not affected the same way with whole blood

■ CONCLUSIONS

A facile, low-energy, two-step coating procedure inspired by mussels was developed to construct first a PDA/PEI layer that strongly adhered to the ePTFE surface, followed by covalent attachment of PGS, which contains a zwitterionic moiety for enhanced biocompatibility. Without shaking, the layers were found to be extremely thin and lightweight, but their existence was confirmed immediately with color changes, and more accurately with the nitrogen and sulfur peaks in the XPS spectra. The effects of PEI molecular weight on hydrophilicity were confirmed in the first coating step: lower molecular weights were more preferred because although the same reactions could have occurred with the same number of reacting groups in PEI, longer chains are more susceptible to intramolecular interactions, effectively burying most of the catechol groups from PDA that are responsible for adhesion to the ePTFE surface. Despite these differences, hydrophilicity was enhanced in all cases, especially after the second coating step. It has turned out that while all PGS-coated membranes performed very similarly due to the saturating effect of zwitterion-water interactions at the surface, surface morphology had a significant contribution when the membranes came into contact with whole blood samples due to higher concentrations of blood plasma proteins. The extent of prewetting during PBS incubation also had a significant effect, with prewetted membranes allowing greater access to interior pores by the fouling solutions. These results provide a basis for further studies using the developed modification strategy with 600 Da PEI. It will be interesting to study the behavior of the membranes with respect to tissue adhesion, which generally prefers larger pore sizes, and cytotoxicity in vitro, which could possibly be used to determine an optimum set of coating parameters and further determine the modification's overall effectiveness for grafted materials.

■ ASSOCIATED CONTENT

* sı Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.0c09073.

Confocal laser scanning microscopy images of the bacterial and blood cell attachment experiments; representative confocal laser scanning microscope images for bacterial attachment experiment (Figure S1); representative confocal laser scanning microscope images for blood cell attachment experiment (Figure S2) (PDF)

Scheme 1 .

 1 Scheme 1. Schematic Representation of the Overall Zwitterionization Procedure

Figure 1 .

 1 Figure 1. Coating density measurements and actual color changes seen for ePTFE membranes subjected to the two-step coating procedure described. The average values of six independent samples are presented.

Figure 2 .

 2 Figure 2. SEM micrographs showing the surface and cross section (inset images) of the virgin and coated samples.

  https://dx.doi.org/10.1021/acsami.0c09073

Figure 3 .

 3 Figure 3. XPS analysis (C 1s, N 1s, and S 2p spectra) showing evidence for successful grafting of PDA/PEI and PGS layers onto ePTFE.

Figure 4 .

 4 Figure 4. Hydrophilicity of the virgin ePTFE and coated membranes shown through (a) dynamic water contact angle measurements and (b) the average hydration capacity of six independent measurements via immersion in water with their standard deviations as error bars.

Figure 5 .

 5 Figure 5. Bacterial attachment experiment for 24 h of E. coli (Gramnegative) and S. aureus (Gram-positive). Relative bacterial coverage (normalized using virgin ePTFE sample) was calculated using ImageJ software. The average values of nine CLSM images are presented with their standard deviation as error bars.

Figure 6 .

 6 Figure 6. Hemocompatibility tests on modified PTFE membranes with human whole blood and RBC-rich fraction. Relative blood cell attachment coverage (normalized using virgin ePTFE sample) was calculated using ImageJ software. The average values of nine CLSM images are presented with their standard deviation as error bars.

Table 1 .

 1 Porosity of Virgin and Coated Membranes

	sample ID	porosity (%)
	virgin ePTFE	45.05 ± 0.42
	D-P600	45.41 ± 0.16
	D-P1800	45.43 ± 0.48
	D-P10000	45.20 ± 0.44
	D-P600-GS	43.89 ± 0.49
	D-P1800-GS	44.15 ± 0.35
	D-P10000-GS	44.43 ± 0.49
	Scheme 2. Proposed Chemistry of the Coating Procedure

https://dx.doi.org/10.1021/acsami.0c09073

Table 2 .

 2 Elemental Composition of Samples Obtained via XPS

			sample composition (atomic %)
	element	virgin	D-P600	D-P600-GS
	C	33.88	51.59	52.43
	F	66.12	38.49	33.14
	N		3.46	1.84
	O		6.46	10.95
	S			1.65

https://dx.doi.org/10.1021/acsami.0c09073

https://dx.doi.org/10.1021/acsami.0c09073

The authors acknowledge the projects of Distinguished Professorship at Chung Yuan Christian University, Ministry of Science and Technology and the Agence Nationale de la Recherche (MOST-ANR International Program: MOST 109-2923-E-033-001), and the National Science Foundation (NSF 1806138) for their financial support. P.M.P.T.F. extends his gratitude for the financial support from the Department of Science and Technology-Science Education Institute (DOST-SEI) and Engineering Research and Development for https://dx.doi.org/10.1021/acsami.0c09073

Technology (ERDT) of the Philippines. The authors also express their gratitude to Professor Antoine Venault of the Chemical Engineering Department of Chung Yuan Christian University for the guidance and inspiration he provided for this research.

Author Contributions

○

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. P.M.P.T.F. and G.V.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS


domain: chim sde

text:
Cunjun Li

Qiqi Wu

Sabine Petit

Will P Gates

Huimin Yang

Weihua Yu

Chunhui Zhou

Prof. C H Zhou

Insights into the Rheological Behavior of Aqueous Dispersions of Synthetic Saponite: Effects of Saponite Composition and Sodium Polyacrylate

Synthetic saponite (Sap) easily delaminates in water to form a transparent sol and hydrogel with excellent rheological performance and is thus widely used in paints, cosmetics, and nanomaterials. The thixotropic property of Sap hydrogels is heavily dependent on the nature of Sap and the external electrolyte and polyelectrolyte; yet, details on the relationship between rheological behaviors of saponite hydrogels and Sap composition and polyelectrolyte remain unclear. In this work, thixotropic rheological behaviors of a series of synthetic Sap hydrogels, with and without added sodium polyacrylate polyelectrolyte (NaPA), were investigated. The Sap samples, with a Si/Al molar ratio from 5 to 25, were successfully synthesized using hydrothermal methods and characterized by powder X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy. The rheological performances of aqueous Sap dispersions and particle sizes and ζ-potentials of Sap were measured. The results showed that the crystallinity of the Sap increased with an increasing Si/Al molar ratio. All Sap samples, with the exception of the Sap with a Si/Al molar ratio of 5, dispersed well in water (3 wt %) to form hydrogels. The rheological behaviors of the hydrogels were related to the chemical composition and the layer charge of the Sap. The Sap with a Si/Al molar ratio of 25 had higher viscosity due to improved delamination. The addition of the NaPA, an anionic polyelectrolyte, into the hydrogels decreased the viscosity and altered the thixotropic properties such that the hydrogel becomes a sol. The addition of NaPA facilitated the dispersion and delamination of Sap, because under the electric field of negatively charged Sap particles in the hydrogel, the anionic NaPA was instantaneously polarized and thereby entered the hydration layer of the Sap particles.

Introduction

Saponite (Sap) is a 2:1 trioctahedral clay mineral. A layer of Sap is composed of two sheets of hexagonally arrayed [SiO4] tetrahedra sandwiching a central sheet of [MgO4(OH)2] octahedra.

A small amount of isomorphous substitution of Si4+ for Al3+ (and sometimes other trivalent cations such as Fe3+) in the tetrahedral sheet causes a negatively charged sheet. In many types of Sap, Al3+ or Fe3+ can substitute Mg2+ in the octahedral sheet, creating a positive octahedral sheet charge; however, substitution in the tetrahedral results in an overall negative net charge of the 2:1 layer. [START_REF] Newman | The Chemical Composition of Clays[END_REF] The negative layer charge is compensated by exchangeable cations such as Na+ , NH4 + , K+ , Li+ , and Mg2+, which reside in the (generally) hydrated interlayer. An ideal structural formula of Sap is thus described as Mx/z z+ [Mg6][Si8-xAlx]O20(OH)4•nH2O, where Mz+ represents the interlayer cations, x ranges from approximately 0.4-1.2, and n represents the number of surface-and/or cation hydration water molecules.2 Compared to montmorillonite, Sap generally has a smaller particle size and higher thermal stability. [START_REF] Tao | Silylation of saponite with 3-aminopropyltriethoxysilane[END_REF] In particular, Sap can be more readily delaminated and exfoliated into nanoplatelets or even single nanolayers in water. [START_REF] Takagi | Size-Matching Effect on Inorganic Nanosheets: Control of Distance, Alignment, and Orientation of Molecular Adsorption as a Bottom-Up Methodology for Nanomaterials[END_REF] As is typical of high swelling smectites, in aqueous dispersion, its solid-water interfacial properties enable it to form colloidal sols or gels. For this reason, Sap has been used for many decades as a nontoxic thickener,5 plasticizer,6 or emulsifier7 in products such as paint, plaster, cosmetics, personal care products, and adhesives.8 Naturally occurring saponite deposits are scarce and generally contain mineral and metal impurities, and hence laborious and time-consuming purification is required.9 This limitation restricts the use of natural Sap; the potential of its synthetic counterparts in industrial applications is therefore of interest. The technology of synthesizing Sap-like clay minerals with a well-controlled chemical composition has been successfully developed, and the products are generally referred to as a synthetic Sap (Sap). In the past, three successful approaches in the synthesis of Sap have been applied: microwave-assisted,10 nonhydrothermal,11,12 and hydrothermal. [START_REF] Intachai | Hydrothermal synthesis of zinc selenide in smectites[END_REF][START_REF] Suquet | Synthesis and Swelling Properties of Saponites with Increasing Layer Charge[END_REF] The microwave-assisted method is conducive to reduction of synthesis time and control of the structure with large pores and high specific surface area. [START_REF] Gebretsadik | Use of polymer as template in microwave synthesis of saponite. Study of several factors of influence[END_REF][START_REF] Bergada | Microwave effect during aging on the porosity and basic properties of hydrotalcites[END_REF] To make the process rapid and feasible in industrial-scale, the nonhydrothermal method under mild condition was developed.12,17 Typically, a gel containing the source of Si, Al, and Na was first prepared at room temperature, followed by mixing it with a solution of urea and metal salts (introducing special metal cations, such as Co and Ni). In this way, the octahedral sheets of saponites can be easily designed to contain one or more of different metal cations. As the hydrothermal method requires only relatively simple equipment and the reaction conditions are easy to control with precision, it is the frequently used approach for Sap synthesis. [START_REF] Intachai | Hydrothermal synthesis of zinc selenide in smectites[END_REF] For many industrial applications, Sap (whether synthetic or natural) commonly needs surface engineering (functionalization), such as intercalation and/or hybridization to enhance its reactivity. Recently, various new Sap-based functional materials, such as nanoparticles/Sap hydrogels, surface-functionalized Sap, and inorganically modified and hybridized Sap, were designed to produce new functional materials. These Sapbased functional materials may exhibit exceptional biological, electrical, and optical properties and applied in electromagnetic, magnetorheological, catalytic, and biological fields.18-20 Such functionalization is usually conducted in aqueous dispersions. In addition, electrolytes are frequently added to adjust the rheological performance of the Sap-based products in practice. Therefore, understanding the physical and chemical interactions between Sap and electrolytes in an aqueous dispersion is critical and fundamental. Deep experimental and theoretical understanding of the rheological behavior of aqueous dispersions of synthetic saponite is worthwhile. Aqueous dispersions and hydrogels of Sap can exhibit different physical states, for example, stable colloidal dispersion or viscoelastic sol and elastic solids. [START_REF] Annan | Clay mixtures and the mechanical properties of microporous and nanoporous ceramic water filters[END_REF][START_REF] Tao | Hydrothermal transformation of mixed metal oxides and silicate anions to phyllosilicate under highly alkaline conditions[END_REF] The rheological properties of Sap hydrogels are related to electrolyte concentration.23 Shear thinning is a common phenomenon for flocs/aggregates that break down when shear is applied. However, shear thickening is uncommon for most clay dispersions and implies that energy input is dissipated into enhanced gels. Thixotropy is the hysteresis in shear stress between increasing and decreasing shear rate paths. Most clay gels undergo shear thinning with increasing shear rate but some thickening when the shear rate decreases after a prolonged exposure to a high shear rate. The Sap hydrogel system, at concentrations less than 1.1 wt % of solid content, displays a typical Newtonian flow, but at concentrations exceeding 1.2 wt %, it can exhibit distinct shear thinning.24 Aspects of how the chemical composition and crystallinity of Sap affect the complex rheological behavior of Sap hydrogels remain elusive. For the purpose of tuning the shear thinning of Sap dispersions for different applications, electrolytes or polyelectrolytes can generally be introduced to help dispersion,25 stabilization,26 or flocculation of clay mineral suspensions.27 Sodium polyacrylate (NaPA, denoted SN-5040 in the industry) is one of the most frequently used polyelectrolytes in paints, pigments, and waste water treatment.28,29 Therefore, the thixotropy of Sap dispersion is a major factor affecting the workability of these products. However, details regarding thixotropic interactions with Sap in water have been rarely reported and rheological behaviors of aqueous Sap-NaPA dispersions are unknown. The objective of this work is to correlate the rheological behavior of Sap hydrogels as a function of the chemical composition of Sap and load rates of NaPA.

2.Experimental Section

Synthesis of saponite

The synthesis procedure was as follows: a mixture of 36.3 g of NaOH and 65.6 g of NaHCO3 was dissolved in 500 mL of deionized water to develop solution A. Then, 88.83 g of sodium silicate was added in 490 mL of solution A under vigorous stirring to create solution B. A given amount of MgCl2•6H2O and AlCl3•6H2O was dissolved in 175 mL of deionized water (solution C) to provide a range of Si/Al in the final product. Under continuous stirring, solution C was slowly added into solution B until a uniform white suspension was observed. The suspension was then transferred to a polytetrafluoroethylene-lined autoclave and heated at 200 °C for 24 h. The solid phase was washed with deionized water to eliminate chlorides and finally dried at 90 °C in an oven and ground in a mortar. The obtained product was denoted Sapx, where x represents the Si/Al molar ratio. For example, Sap5 means the Sap synthesized at the Si/Al molar ratio of 5.

Preparation of Sap Hydrogels and NaPA-Loaded Sap Dispersion

Sapx (2.47 g) was added into 80 mL of deionized water, and the mixture was stirred to form a suspension (3 wt % Sap). A total of 30 mL of suspension was then transferred to a tube, and the remaining 50 mL of suspension was transferred and divided into five tubes with an equal volume of 10 mL. After standing for 7 d at room temperature, Sap hydrogels were obtained. For the preparation of NaPA-loaded Sap dispersion, 0.25, 0.50, 0.75, 1.0, and 1.25 mL NaPA solution (4 g/L) was added into the above five 10 mL tubes separately. The corresponding mixtures of different concentrations were denoted Sapx-NaPAy, where x represents the Si/Al ratio of the dispersed Sap at 5, 10, 15, 20, or 25 and y is the concentration of added NaPA at 0.10, 0.19, 0.28, 0.36, or 0.44 g/L aqueous Sap dispersion. For example, Sap20-NaPA0.44 indicates that the mixture was made by Sap20 with a concentration of NaPA at 0.44 g/L. To study the structure of the Sap after dispersion in the presence of NaPA, Sapx-NaPA0.44 (the concentration of NaPA in Sapx dispersion is 0.44 g/L) was dried at 80 °C to a constant weight. For comparison, Sap dispersions without adding NaPA were dried under the same conditions and used as a control. Such a sample was denoted Sapxcontrol.

Powder X-ray Diffraction (XRD) and X-ray Fluorescence (XRF)

Powder X-ray diffraction (XRD) analysis was carried out on an X-ray diffractometer (X'Pert PRO, PANalytical, The Netherlands). Cu target Kα (λ = 0.154056 nm), a voltage of 40 kV, a current of 40 mA, and a scanning range of 2-80° were used. Elemental analyses of the samples (pressed powders) were carried out using an ARL ADVANT'X intelliPowertTM 4200 sequential X-ray fluorescence (XRF) analyzer. The layer charge density calculation of the structural formula was based on well-known assumptions about the structure of Sap (2:1 layer structure, 22 oxygen equivalence, all Mg assigned to an octahedral sheet, etc.).30

Fourier infrared spectroscopy (FTIR)

Fourier transform infrared spectra (FTIR) were recorded in transmission mode with a Nicolet 6700 FTIR spectrometer (Thermoelectric Nicolet, United States). Pressed disk samples were prepared by mixing the powdered solids with KBr in a pelleting device. All samples were measured at a wavenumber range of 400-4000 cm-1 with 32 scans. The wavenumber accuracy was better than 0.01 cm-1 , and the resolution was better than 0.09 cm-1.

Pore Structure Characterization

The pore size distribution of Sap was detected on a Micromeritics ASAP2020 analyzer. Before the measurements, the Sap samples were degassed at 473 K for 10 h under a N2 flow.

Scanning Electron Microscopy (SEM)

For SEM characterization, the powder sample was mounted onto a conductive adhesive, coated in gold, and then analyzed on a Hitachi S-4700(II) scanning electron microscope.

Rheological measurement

The rheological properties of the Sap hydrogels were measured using a Malvern Kinexus Rheometer (Malvern Kinexus Lab+ Rheometer). The flow measurements were carried out at 25 ± 0.1 °C using a rotational rheometer with a CP4/ 40 SR71SS cone-plate sensor; oscillatory measurements were carried out over a frequency range of 0.1-10 Hz at 25 ± 0.1 °C using a stress control rheometer equipped with a CP4/40 SR71SS cone-plate sensor system. The data was analyzed using the rSpace software for the Kinexus rheometer.

Zeta potential and dynamic light scattering (DLS) measurement

. A fresh Sap dispersion was prepared by adding 1.22 g of Sap into 80 mL of deionized water. The zeta potential and particle size distribution of the synthetic saponite dispersion were performed with a Malvern Nano ZS90 instrument.

Results and Discussion

Mineralogy of Sap

All synthetic samples (Figure 1c) exhibited reflections at 2θ 6.03, 19.52, 29.04, 34.41, and 60.37°, corresponding to the (001), ( 0211), ( 004), (1320), and (060) reflections of saponite, respectively.4

In particular, the position of the (060) reflection (d060 value) differentiates the structure of Al-enriched dioctahedral and Mg-enriched trioctahedral smectite.31 The d060 value of the series of synthetic samples was 0.153 nm, indicating that the synthesized smectites have a trioctahedral structure (Figure 1a,b). This is in good agreement with the data reported in the literature that the d060 of the trioctahedral structure is higher than 0.152 nm.32,33 As such, in this work, Sap with Si/Al molar ratios of 5, 10, 15, 20, and 25 was successfully created with the hydrothermal process.

The (001) reflections of the samples Sap5, Sap10, Sap15, Sap20, and Sap25 are 6.68, 6.13, 6.03, 5.88, and 5.78°, corresponding to basal spacings of 1.33, 1.44, 1.46, 1.50, and 1.53 nm, respectively. As the Si/Al molar ratio increases from 5 to 25, theoretically, the layer charge decreases from 1.33 to 0.35 e/pfu (Table 1). Thus, the layer charge density decreases. Usually, the basal spacing of smectite is controlled by the attractive and repulsive forces between two adjacent layers. [START_REF] Rajapakse | Preparation and characterization of electronically conducting polypyrrole-montmorillonite nanocomposite and its potential application as a cathode material for oxygen reduction[END_REF] As the layer charge increases, the electrostatic attraction increases more than the repulsive force.8 Hence, the basal spacing followed the order Sap5 < Sap10 < Sap15 < Sap20 < Sap25. As the Si/Al molar ratio increased from 5 to 25, the intensity of the (001) and (004) reflections decreased, whereas their full widths at half-maximum (FWHM) significantly decreased (Figure 1c). A similar trend was also observed for the (060) and (100) reflections. These observations indicated that the synthetic saponites with a lower Si/Al ratio had superior crystallinity than those with a higher Si/Al ratio. Thus, a Si/Al molar ratio in the hydrothermal synthesis feedstock has a strong influence on the crystallinity of the resultant Sap. Decreasing the molar ratio of Si/Al favored Sap with larger crystal size and superior stacking order. As the Si/Al molar ratio inherently contributes to isomorphous substitution in the tetrahedral sheet of the saponite layer, greater content of Al in the tetrahedral sheet leads to superior crystallinity of the synthetic saponite. [START_REF] He | Aluminum ion occupancy in the structure of synthetic saponites: Effect on crystallinity[END_REF] The successful formation of Sap was also confirmed by FTIR spectroscopy (Figure 1d). The characteristic absorption bands36 of Sap were observed at 3685, 1004, 800, 665, and 460 cm-1 . The absorption band at 3685 cm-1 , which arose from the stretching vibrational modes of Mg-OH, is a typical characteristic of trioctahedral clay minerals. [START_REF] Madejova | FTIR techniques in clay mineral studies[END_REF] The broad band centered at 3440 cm-1 and the band at 1645 cm-1 were both due to adsorbed water molecules.

The decrease of the band at 800 cm-1 , due to tetrahedral Al-O vibrations from Sap5 to Sap25, indicated a decrease of the tetrahedral Al content according to the chemical composition of the synthetic saponites (Table 1). The band at 1058 cm-1 reflects the stretching vibration of the Si-O-Si. The slight shift of this band from Sap5 to Sap25 also indicated a decrease of tetrahedral substitutions.38

Rheology of Sap

When a Sap is dispersed in water, it forms a colloid. In particular, the colloids of Sap10, Sap15, Sap20, and Sap25 appeared to be strongly optically transparent. After standing for 7 d, samples Sap10-Sap25 formed hydrogels (Figure S1aI). When the tubes were put upside down, the gels remained in place unless vibration (shear force) was applied. In contrast, Sap5 failed to form a hydrogel. This can be ascribed to the better crystallinity and higher layer charge of Sap5. In addition, the average particle size of the Sap5 is 2-3 times larger than that of the other Sap (Table 1). Such Sap easily sedimented in suspension. The better crystallinity and layer charge make it difficult to delaminate; thus, it failed to form stable dispersions. Due to Tyndall scattering, a bright path in the hydrogels (Figure 2cI) of Sap10, Sap15, Sap20, and Sap25 can be observed. This suggests that the Sap had delaminated in the aqueous dispersion because of the electrostatic repulsion of Sap particles and thus existed as nanoparticles. Only colloids of Sap5 kept the form of a sol and did not form a gel. Thus, the rheological properties of Sap5 were not measured in the following experiments. Under a nonsteady state, the viscosity of the Sap hydrogel (3 wt %) as a function of shear rate (Figure 2a) suggested that the hydrogels showed distinct shear-thinning behaviors. At the lowest shear rate of 0.1 s-1 , the viscosities of the Sap hydrogels of Sap10, Sap15, Sap20, and Sap25 were 132.3, 149.6, 241.8, and 306.2 Pa•s, respectively. As the shear rate increased to 100 s-1 , the viscosity decreased. At a fast decrease from 0.1 to 2.512 s-1 , the viscosity decreased rapidly, and finally, the viscosity reached a minimum and tended to be equal for all four samples. A possible reason for the observed decrease of viscosity with increasing shear rate was that the house-of-card structure, with edge-to-face interactions between Sap nanolayers, was destroyed by shear force,39 causing the direction of the delaminated layer of Sap in the hydrogel to gradually change to the direction of flow under shear force, resulting in the decreasing viscosity. At a shear rate of 100 s-1 , the viscosity of the aqueous Sap dispersions approached zero, revealing a typical shear-thinning process. The solidlike gel thus transformed to liquidlike sol at the highest shear rates. At a given shear rate, Sap samples with a higher layer charge density had lower-viscosity hydrogels. Sap20 and Sap25 resulted in hydrogels with greater swelling. A similar conclusion that greater charge density led to stronger cohesive strength between the layers, thus decreasing swelling, was also drawn in the literature.40 Thus, that the layer charges of 0.53 e/pfu (Sap15) to 0.35 e/pfu (Sap25) resulted in greater swelling is a well-accepted deduction. Generally, because the Sap particles delaminate to isolated mono, or at least partially, delaminated multilayers, the increase in layer charge resulted in less delamination of Sap particles. [START_REF] Chemeda | Rheological properties of palygorskite-bentonite and sepiolite-bentonite mixed clay suspensions[END_REF] Experimental results of shear rate versus viscosity can be fitted to the Sisko42 model (Figure S1a). η = η∞ + kγn-1 , where η∞ denotes the infinite-rate viscosity in Pa•s, k is a constant, n is a rate index, and γ represents shear rate. At a low shear rate, the house-of-card structure of Sap in a hydrogel strongly hinders rotation, thus requiring appreciable torque to initiate flow with the rotational rheometer. However, the house-ofcard structure was destroyed gradually as the shear rate increased. According to the Sisko model, at a shear rate of 100 s -1 , the Sap hydrogel approached infinite viscosity (η∞).

Shear stress, as a function of the shear rate (Figure 2b) of Sap (Sap10, Sap15, Sap20, and Sap25) hydrogels (3 wt %), showed similar behavior to non-Newtonian fluids.43 At a shear rate of 0.1 s-1 , the shear stresses of the hydrogels of Sap10 and Sap15 were 13.24 and 14.96 Pa, whereas the shear stresses of Sap20 and Sap25 were 24.18 and 32.94 Pa, respectively. The data indicated that shear stress is closely related to the layer charge density of Sap. The shear stress increased with the decrease of layer charge density from Sap10 to Sap25. Inherently, higher shear stress means that the particle-particle associations require higher energy to break and/or a faster rate of bond formation over shear-initiated breakage. These interactions are decided by the layer charge density and particle size of Sap. In this context, according to σ = EK(N2 /2) (where σ is shear stress, E is the energy of the bond, K is a rate constant for bond formation and breakage, and N is the number of particles per cm3 that are constantly forming and breaking bonds during the shear/flow process),44 and provided that the bond energy between Sap particles and the rates at which bonds form and break are almost constant in the hydrogel of the Sap20 and the Sap25, the number of bonds forming and breaking will play a larger role in shear stress than that in the Sap10 and the Sap15. Since all dispersions were completed at the same solid concentration, any changes in the number of bonds forming or breaking can be attributed to differences in particle size (number of layers associated with each quasicrystal). The number of layers associated with each quasicrystal can be then related to layer charge as discussed above. Furthermore, as (plastic) viscosity is represented by the slope of the shear stress curve, σ = σe + ηΩ (where σe is the extrapolated shear stress, η is viscosity, and Ω is the shear rate),45 viscosity is also influenced strongly by N. It worth noting that at a shear rate of 1.45 s-1 , Sap20 and Sap25 followed a typical plastic flow, whereas dilatant flow behavior was observed for Sap10 and Sap15. The phenomena implied that, given the higher layer charges for Sap10 and Sap15 resulting in larger particles, above a threshold shear force, delamination of Sap particles occurred and thus increased swelling resulted in increased dispersion viscosity. Experimental results of shear rate versus shear stress can be fitted to the Herschel-Bulkley model.46 τ = τ0 + kγn , where τ0, k, and n are the Herschel-Bulkley yield stress, the consistency index, and the flow index parameters, respectively. The model also indicates that, under a certain shear force, delamination of Sap10 and Sap15 layers occurred and thus led to increased swelling when dispersed. The dispersion of Sap10 and Sap15 flowed because of their higher layer charge and larger particles. Regarding Sap20 and Sap25, both exhibited plastic or Bingham flowing behavior. A linear response above the critical shear rate means that the rate of energy dissipation per unit volume is at equilibrium with the rate of shear. By contrast, for Sap10 and Sap15 dispersions, only the rate of energy dissipation per unit volume is at equilibrium with a shear rate of 1.45 s-1 . Under a nonsteady state, the Sap hydrogels displayed thixotropy (Figure 2c), as the plots of shear stress versus shear rate exhibited characteristic hysteresis47 and revealed that all Sap hydrogels are non-Newtonian fluids. Thixotropy is defined as the area within the flow curve loop, increasing and then decreasing with the applied shear rate. Thixotropy values for Sap10 and Sap15 hydrogels were 4830 and 4643 Pa•s -1 , whereas for Sap20 and Sap25 hydrogels were 10573 and 7706 Pa•s -1 , respectively, in the shear rate range of 1.45-1000 s-1 . Sap20 had the largest thixotropic loop area and also displayed the highest shear stress. The larger area of the thixotropic loop for Sap20 and Sap25 hydrogels revealed that they exhibited a stronger thixotropy than Sap10 and Sap15 hydrogels. Such differences in thixotropy might be caused by the distinct particle associations in sample Sap20 as observed by SEM (Figure 7g). Theoretically, in the Sap gel, equilibrium is never achieved between the bond formation and bond breakage, as the Sap system undergoes shear. In regard to Sap10 and Sap15 hydrogels, the lack of oscillations indicates that equilibrium conditions are essentially met. Some further evidence of thixotropic behavior being affected by layer charge density and particle size (inherently particle associations and bonding) can be seen as oscillations in the shear responses within the hysteresis loops (Figure 2d). The storage modulus (G′) reflects the energy storage and elastic properties of the material after perturbation. [START_REF] Gao | Source apportionment of atmospheric PAHs and their toxicity using PMF: impact of gas/particle partitioning[END_REF] The loss modulus (G″) reflects the energy loss and viscous properties of the material through the relaxation or dissipation of energy (usually as heat).49 For the four samples, the G′ exceeded the G″ at a frequency ranging from 1 to 10 Hz at 25 ± 0.1 °C and indicated that the Sap dispersions behave as hydrogels. [START_REF] Zamora-Mora | Magnetic core -shell chitosan nanoparticles: Rheological characterization and hyperthermia application[END_REF] In other words, they tended to form bonds and decrease N over time. The hydrogel formed by the Sap20 possessed the highest G′ and G″.

Crystallinity and morphology of Sap

All of the Sap samples exhibited a plated morphological structure as observed in SEM images (Figure 7). For the SEM images of Sap5 (Figure S7), slices of curved plates with well-defined edges can be clearly observed, indicating that well-crystallized layered saponite was achieved.34 Compared to Sap5, the Sap10 samples consist of smaller secondary and primary Sap particles (Figure 7a). Thus, incorporation of less Al into the Sap framework hampered the formation of large plates. This trend was more obvious for the Sap15 (Figure 7b). In particular, Sap20 (Figure 7c) has a larger size than the other Sap, except for Sap5, possibly a result of distinct aggregation. This distinction has been found in the measurement of the thixotropic behavior of the gel from aqueous Sap20 dispersion (Figure 5). The SEM image of the Sap25 (Figure 7d) also showed a platy morphology. The results (Table 1) obtained from dynamic light scattering (DLS) also revealed that Sap20 behaved differently when compared with the others. The average particle sizes of Sap5, Sap10, Sap15, Sap20, and Sap25, as measured by DLS, were 502, 171, 148, 245, and 144 nm, respectively. Particle size decreased gradually with an increasing Si/Al molar ratio from 5 to 15 but increased by ca. 100 nm for Sap20. These results correlate well with SEM observations and rheology results. Moreover, the pore structure of Sap20 appeared different. The average pore diameters of Sap5, Sap10, Sap15, Sap20, and Sap25 were 3.61, 3.68, 3.34, 3.08, and 3.16 nm, respectively (Figure S8). The Barrett-Joyner-Halenda desorption cumulative pore volumes of Sap5, Sap10, Sap15, Sap20, and Sap25 between 2.05 and 3.10 nm were 0.0122, 0.0118, 0.0416, 0.0624, and 0.0490 cm3 /g, respectively. The differences in particle size and pore structure are related to the formed hydrogel. The hydrogel strength (selling pressure) is much stronger in Sap20 than the others. In Sap-water systems, an electronic double layer was made up of the negative surface charge and the balancing cation charge. As discussed above, for all Sap samples, the negative charge of the Sap layer is a consequence of isomorphous substitutions within the saponite crystal lattice. The counterions were electrostatically attracted by the oppositely charged surface (Figure 2e) but had a tendency to diffuse away from the surface to the bulk solution where its concentration is lower. The concentration of the counterions near the Sap particle surface was high, and it decreased with increasing distance away from the surface of the Sap particle. The diffuse layer did not only consist of an excess of opposite ions but also a deficiency of anions near the surface, as the ions with the same charge were electrostatically repelled by the surface of the Sap particle. Here, in the sap-water suspension system, the cations were adsorbed onto the surface of Sap particles and, together with the counterions, formed a diffuse double layer (Figure 2e). The higher charge potential at the Sap surface overrode the tendency for the cations to hydrate with water, so the charge was more effectively neutralized and the diffuse double layer contracted. As such, the (absolute value of) ζ-potential of Sap dispersion also appeared to be higher for the Sap carrying a higher layer charge density (Figure 5a). The gel-forming ability of the dispersion of Sap in water was affected by both the layer charge density of Sap and the relevant associations and connections of Sap particles. For the lowest layer charge density, Sap can be more easily dispersed and delaminated in water to form a house-of-card structure. Such gels displayed higher viscosity and thixotropy, explaining why the Sap20 hydrogel exhibited the largest area of a thixotropic loop (Figure 2c) and the Sap25 hydrogel exhibited the highest viscosity. This implies that the thixotropic rheological properties of the Sap gel were closely related to the chemical composition of Sap and association modes between the Sap particles into water.

Rheological behavior affected by sodium polyacrylate

For the purpose of tuning the shear thinning of Sap hydrogels and studying the influence of the NaPA on the different Sap hydrogels, NaPA with concentrations of 0.1, 0.19, 0.28, 0.36, and 0.44 g/L was loaded into the Sap hydrogels. The infinite viscosity of the Sap-NaPA dispersions tends to zero. This phenomenon indicated that the addition of NaPA induced shear thinning (Figure 3a-d).

In regard to Sap-NaPA0.1 dispersions made from Sap10, Sap15, Sap20, and Sap25 hydrogels with added NaPA at a concentration of 0.1 g/L, the viscosity decreased rapidly as the shear rate increased from 0 to 2.5 s-1 . Comparing the Sap10-NaPA0.1 dispersion to dispersions of Sap15-NaPA0.1, Sap20-NaPA0.1, and Sap25-NaPA0.1, the decline in viscosity was much slower and may be due to the higher layer charge density of Sap10 (0.74 e/pfu). For all SapNaPA dispersions, as the shear rate increased from 2.5 to 10 s -1 , the viscosity declined slowly. When the shear rate increased to more than 10 s-1 , all Sap-NaPA dispersions were similar to liquids with very low viscosity, especially at the equilibrium viscosity. The result suggested that the effect of NaPA on Sap hydrogels was less significant at very high mechanical forces and the addition of NaPA weakened the particle-to-particle connections between Sap particles in hydrogels.

Furthermore, the Sap dispersions in the presence of NaPA are well fitted with the Sisko model (Figure S2a-d). The infinite viscosity of the Sap-NaPA dispersions tends to zero, indicating that the addition of NaPA induced shear thinning. Shear stress as a function of shear rate for different SapNaPA dispersions is shown in Figure 4. The results of shear stress versus shear rate showed that the Sap-NaPA dispersion followed a plastic flow and fit the Herschel-Bulkley τ = τ0 + kγn model46 (Figure S3a-d). The shear stress of all Sap-NaPA dispersions decreased with the increase of the concentration of the NaPA (Figure 4a-d). In addition, it was noted that the shear stress increased with increasing shear rate, except for Sap10-NaPA0.1, in which the shear rate had an indistinctive influence on shear stress (Figure S3a). The reason is that the layer charge density of Sap10 (0.74 e/pfu) is higher than the others. The low concentration (0.1 g/L) of NaPA had an insignificant influence on the hydrogel formation for Sap10.

Without adding NaPA in Sap hydrogels, the yield stresses (τ0) of Sap10 and Sap15 were 13.35 and 14.93 Pa, respectively, whereas they were 29.33 and 66.17 Pa for Sap20 and Sap25, respectively.

When the concentration of added NaPA was 0.1 g/L, the yield stresses (τ0) of Sap10-NaPA0.1 and Sap15-NaPA0.1 were 7.09 and 7.38 Pa, respectively, whereas they were 13.32 and 25.65 Pa for Sap20-NaPA0.1 and Sap25-NaPA0.1, respectively. With an increased NaPA concentration, the yield stress τ0 decreased gradually and tended toward zero, indicating that the Sap hydrogel tended to be sol when NaPA was added. The Sap20 and Sap25, with a lower layer charge density than Sap10 and Sap15, were more difficult to flow for their hydrogels and dispersions with addition of NaPA. The thixotropic behavior of Sap dispersions was also affected by NaPA. For all Sap-NaPA dispersions, the dispersions are still thixotropic (Figure S4a-d). NaPA concentration had no observable effect on the area of the thixotropic loop for Sap20-NaPa, but the areas of the Sap20-NaPA complexes were reduced significantly compared to that of the Sap20 hydrogel. The same phenomenon was observed for Sap25-NaPA dispersion. Such results in thixotropy suggested that the concentrations of NaPA had an indistinctive influence on thixotropy for Sap20 and Sap25 hydrogels. The shear thinning and thixotropy of Sap dispersions derived from the characteristics of the network formed by Sap particles; the network was destroyed as the shear rate increased. In contrast, when the shear rate decreased, the aggregation of Sap layers could be rebuilt to be a whole network. The frequency response of the G′ and the G″ of the Sap dispersions in the presence of NaPA is shown in Figure S5a-d.

For all Sap-NaPA dispersions, as the frequency increased, G′ was first lower than G″ and then G′ became higher than G″. Compared to Sap dispersions without the addition of NaPA, the G′ at the analyzed frequencies decreased by several orders of magnitude and the G″ was higher than the G′ at lower frequencies. In other words, adding NaPA in the Sap dispersion led to a viscoelastic liquidlike sol.51 This sol was not a pure viscous fluid at the analyzed frequencies. The elastic component of the viscoelastic sol indicated a tendency to gel.52 Based on the discussion of the above results, it can be concluded that the addition of NaPA induced changes in the rheological properties of Sap hydrogels. After adding NaPA into the Sap hydrogel system, the Sap hydrogels transformed to sols by weakening the connections between Sap particles, resulting in a decrease in the viscosity and thixotropy of Sap dispersions.

Interaction between the anionic polyelectrolyte and Sap

The ζ-potential of 1.5 wt % Sap dispersions was plotted as a function of NaPA concentration (Figure 5a). The ζpotential of all Sap samples is less than -38 mV. Sap with a higher layer charge with addition of NaPA changed, which can be described as the Hofmeister effect. [START_REF] Kunz | The present state of affairs with Hofmeister effects[END_REF] The FTIR spectra of the dried samples, which were made from the Sap dispersions with an added 0.44 g/L NaPA, are similar to those of the samples without added NaPA (Figure S6), except that the presence of NaPA could be observed in the Sap25-NaPA0.44 sample (Figure S6d). The bonds at 1559 and 1325 cm-1 are ascribed to the symmetrical stretching vibrations of COO-. The bond at Sap25-NaPA0.44. Notably, no change in the vibration bands could be observed, revealing that the addition of NaPA did not generate new chemical bonds. The presence of NaPA in the Sap25-NaPA0.44 is probably the adsorption of NaPA on the surfaces of Sap25 by electrostatic attraction and hydrogen bonding. To better illustrate the interaction between NaPA and Sap, a possible schematic illustration is proposed (Figure 5). NaPA has a carboxylate functional group, which ionizes in water (Figure 5bI); thus, it can be dissociated to form PA-. Due to the surface charge in the vicinity of the Sap particle, an electric field strength formed.55 Under such an electric field, the negatively charged PA-underwent an instantaneous polarization,56 which can enter into the Sap hydration sphere faster than the original hydrated ions. PA-would electrostatically interact with the exchange cations as PA-and can approach closer to the cations than the cations to the surface of the clay (due to water). PA-displaced water and formed a strong bond because of the stronger electrostatic attraction. At these very low NaPA concentrations, the PA-interacts with the exterior surfaces of the Sap quasicrystals but eventually by mass action enables delamination. Consequently, after adding NaPA into the Sap hydrogel, the Sap hydrogel transformed into a Sap dispersion resulting from the destruction of the original network structure (Figure 5bII). The destruction of the network structure can be ascribed to the electrostatic attraction between polarized PA-and negatively charged Sap particles and possible hydrogen bonding between broken bonds, Mg-O and Si-O, at the edges of tetrahedral/octahedral sheets of Sap and hydrogen atoms on PA-. Another possible reason is when NaPA was introduced into the Sap hydrogel system, the width of the diffuse layer increased because PA-has a larger molecular size than original hydrate ions. Additionally, to further investigate the interaction between Sap and NaPA, powder XRD and SEM were carried out for Sap-control (dried from the Sap hydrogel) and Sap-NaPA (dried from the Sap hydrogel after adding NaPA). Comparing XRD pattern of Sap10-NaPA0.44 (dried from Sap10 dispersions, after adding NaPA at the concentration of 0.44 g/L) with that of the Sap10-control, the intensity of the (001) reflection increased and the full widths at half-maximum (FWHM) significantly decreased for Sap10-NaPA0.44 samples (Figure 6a). Such results can indicate that the addition of NaPA induced a better coherency in the c dimension for Sap10 layers, i.e., a higher number of Sap layers stacked. Regarding the other SapNaPA0.44 (dried from Sapx dispersions, where x is 15, 20, or 25 mole% Al in Sap, after adding NaPA at the concentration of 0.44 g/L), the intensity of the (001) reflection increased but the FWHM significantly increased when compared with their corresponding Sap-control (Figure 6b-d). The results indicate that PA-was able to penetrate the interlayer, resulting in delamination.

The SEM observations of Sap-NaPA0.44 revealed that these Sap samples have a more compact and ordered layer structure than the raw Sap (Figure 7). Sap in the water system dispersed and formed hydrogels after standing for 7 d and dried from dispersions reassembled in the first instance (Figure 7e-h). A schematic illustration of Sap dispersion with/without added NaPA and reaggregation after drying is presented in Figure 5c to clarify the interaction process between Sap, water, and NaPA. After adding NaPA into the Sap hydrogel, the house-ofcard structure was destroyed and the Sap hydrogel became more dispersed (Figure 5cVI). When the Sap-NaPA0.44 dispersion was dried, the dispersed Sap particles reassembled in a more ordered, regular, and compact structure (Figure 5cVII). Interestingly, as seen in Figure 7g, Sap20-NaPA0.44 exhibited the best compact structure among the Sap-NaPA0.44 samples, suggesting that the reassembly/reaggregation of negatively charged Sap layers reacted with polarized PA-, which may be caused by the strength of the Sap layer charge density. The Sap20 with the layer charge density of 0.42 e/pfu delaminated best in a water/NaPA system and reassembled/ reaggregated better than the other Sap with unmatched layer charge density. Such results further confirmed that the NaPA not only affected the rheological properties of Sap dispersion but also promoted the reassembled Sap to form a superior crystal structure by interacting with the Sap hydration sphere in Sap dispersion.

Conclusion

Saponites, with a Si/Al molar ratio ranging from 5 to 25, were successfully synthesized by a simple hydrothermal process. The Si/Al molar ratio had a remarkable influence on the crystallinity, morphology, and particle size of the synthetic Sap. The lowest Si/Al molar ratio (5) led to improved Sap crystallinity. However, for this lowest ratio, the synthetic Sap did not delaminate easily, probably due to a high layer charge, and was unable to form gel in aqueous dispersion. On the other hand, the aqueous dispersions (3 wt %) of synthetic Sap, with a Si/Al molar ratio ranging from 10 to 25, formed gel spontaneously. The Sap hydrogel had remarkable rheological properties that were related to the chemical composition and inherently to its crystallinity, layer charge, and particle size. The hydrogel from the Sap with a Si/Al molar ratio of 25 exhibited the highest viscosity, which was ascribed to better delamination and the formation of an open house-of-card structure with stronger connections of delaminated Sap particles compared to the other synthetic Sap. The addition of the anionic polyelectrolyte NaPA decreased the viscosity and reduced the thixotropic properties of the Sap hydrogel. Moreover, the addition of NaPA caused the hydrogel to become sol. The aqueous dispersion of the Sap with NaPA formed a stable sol, compared to the Sap sol or Sap hydrogel. The colloidal stabilization of Sap by NaPA is considered to be due to the Hofmeister effect. In the hydrogel, electrostatic interactions between synthetic Sap particles and NaPA occurred. The anionic NaPA could be instantaneously polarized under the electric field of Sap particles and, as a result, quickly entered the hydration layer of Sap particles to disrupt the structure of Sap gel, thereby changing the Sap dispersion in water. Such effects of sodium polyacrylate can be further investigated to finely tune the rheological behaviors of synthetic saponite hydrogels and can be used to exfoliate swelling clay minerals into 2D nanosheets.

  displayed a higher ζ-potential. At a given NaPA concentration, the ζ-potential of Sap10 (0.74 e/pfu) was the highest compared to the other Sap samples. The ζpotential of the Sap represented the change in the layer charge of the particles as a function of their composition. Over the entire range of NaPA concentrations, the ζ-potential values indicated a strong negative charge on the surface of Sap particles. The ζ-potential of negative charge increased significantly as the NaPA gradually covered the surface of the particles. Thus, NaPA increased electrostatic repulsion between Sap particles and promoted the stabilization of Sap dispersion.53 The rheological properties of Sap

Acknowledgments

The authors wish to acknowledge the financial support from the National Natural Scientific Foundation of China (41672033; 21373185), the financial support by the open fund from Key Laboratory of High Efficient Processing of Bamboo of Zhejiang Province, China National Bamboo Research Center,,Key Laboratory of Clay Minerals of Ministry of Land and Resources of the People's Republic of China, Engineering Research Center of Non-metallic Minerals of Zhejiang Province, Zhejiang Institute of Geology and Mineral Resource, China (ZD2018K05), and the State Key Laboratory Breeding Base of Green Chemistry-Preparation Technology, Zhejiang University of Technology (GCTKF2014006).