Biomimicking Dual-Responsive Extracellular Matrix Restoring

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Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Biomimicking Dual-Responsive Extracellular Matrix Restoring Extracellular Balance through the Na/K-ATPase Pathway Xingkun Luan,†,‡ Haozheng Wang,†,‡ Zehong Xiang,† Zhifang Ma,† Jiruo Zhao,‡ Ying Feng,*,‡ Qiang Shi,*,†,§ and Jinghua Yin† †

Downloaded by UNIV OF SOUTHERN INDIANA at 07:01:59:309 on May 31, 2019 from https://pubs.acs.org/doi/10.1021/acsami.9b05420.

State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, Jilin 130022, China ‡ Key Laboratory of Olefin Catalysis and Polymerization/Key Laboratory of Rubber-Plastics (QUST) of Shandong Provincial, Qingdao University of Science and Technology, Qingdao 266042, P. R. China § University of Science and Technology of China, Hefei, Anhui 230026, P. R. China S Supporting Information *

ABSTRACT: Biomedical implant mimicking the physiological extracellular matrix (ECM) is a new strategy to modulate the cell microenvironment to improve implant integrity and longevity. However, the biomimicking ECM suffers from low sensitivity to pathological change and low efficiency to restore the physiological state in vivo. To overcome these problems, reactive oxygen species (ROS) and K+ dual-responsive micro-/nanofibers that encapsulate ascorbic acid-2-glucoside (AA-2G) are fabricated on an elastomer substrate with electrospinning to mimic the ECM. The strategy is based on the fact that ROS and K+ dual responsiveness enhance the sensitivity of the ECM to pathological changes and delivery of AA-2G from the ECM to cell membrane promotes reactivating Na/K-ATPase and shifting cellular diseased conditions to the normal state. We demonstrate that the ROS and K+-responsive tripolymer of poly(ethylene glycol)diacrylate, 1,2-ethanedithiol, and 4nitrobenzo-18-crown-6-ether (PEGDA−EDT−BCAm) are synthesized successfully; the ECM composed of acylated poly(caprolactone)/PEGDA−EDT−BCAm/AA-2G micro-/ nanofibers is prepared through reactive electrospinning; the ECM is sensitive to ROS and K+ concentration in the microenvironment to release AA-2G, which targets the membrane to remove the excessive ROS and reactivate Na/K-ATPase; as a result, the ECM reduces oxidative stress and restores the extracellular physiological state both in vitro and in vivo. This work provides basic principles to design an implant that can adjust the extracellular microenvironment while avoiding pathogenicity to improve implant integrity and longevity in vivo. KEYWORDS: biomimicry, dual-responsive, extracellular matrix, Na/K-ATPase

1. INTRODUCTION The materials that are implanted into living organs or tissues often initiate a host response, such as protein adsorption, cell adhesion, and immune cell activation.1,2 The host immune response leads to an abnormal extracellular environment, which will cause injury to the cells and slow the healing process. However, host immune response cannot be avoided completely and the desired host response supports the healing process.3 For example, inflammation often leads to the overproduction of reactive oxygen species (ROS), which include a large number of chemical species, such as hypochlorite, hydrogen peroxide, superoxide, and hydroxyl anions.4−7 Overproduction of ROS not only induces oxidative damage to the cell but also takes part in plenty of pathological conditions including neurodegenerative and cardiovascular diseases and cancer.8,9 However, ROS below the certain level plays key roles in combating pathogens, modulating cellular signaling pathways, and causing biomaterial degradation.7,10 Therefore, modern implant is designed to restore the extracellular balance after host response in order to improve © XXXX American Chemical Society

implant integrity and longevity while avoiding loss of the intended function by foreign body reactions and chronic inflammation.3 Biomaterials that mimic the physiological extracellular matrix (ECM) prove to regulate the extracellular microenvironment in maintaining cell functions potentially. The ECM is a fibrous structure that mainly contains collagens, proteoglycans, glycoproteins, and smaller amounts of other proteins.11,12 In addition to acting as a scaffold for cells’ organization into tissues, the ECM provides a dynamic microenvironment that perpetually provides physical and chemical signals to the inside cells, serving as a multifunctional regulator of cellular behavior.13 The biomimicking ECM has been fabricated with various materials including ECM proteins, polysaccharides, and synthetic polymers.11−13 Despite the success in regulating cell spreading and biochemical ligand Received: March 27, 2019 Accepted: May 17, 2019 Published: May 22, 2019 A

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

to design implant biomaterials that mimic the ECM to make materials self-adaptive for next generation.

presentation, a biomimicking ECM that can restore extracellular balance is still lacking. The challenge which remains is the low sensibility to pathological change to remove excessive toxic substances and inappropriate target pathway to cell membrane to activate the cellular defense system. Pathological change caused by an implant is often accompanied with ROS overproduction and consequent ion imbalance.14−16 Because excessive ROS and imbalanced ions work together to deteriorate cellular defense systems, the design of the biomimicking ECM that releases biomolecules in response to both ion and ROS is expected to enhance ECM sensitivity substantially. Furthermore, the released biomolecules that not only remove the extracellular toxic substances but also facilitate activation of the cellular defense system by targeting the cell membrane are highly desired. Considering the role in manipulating ROS and ion homeostasis in vivo, Na/ K-ATPase in the cell membrane is an ideal target. Na/KATPase is a transmembrane enzyme for electrogenic ion transportation, responsible for high K + and low Na + concentration in the cytoplasm. Because it is a cellular oxidative stress target, Na/K-ATPase activity is readily inhibited by the excessive ROS,14,15 and Na/K-ATPase deficiency induces increase in K+ efflux and delay in rectifier K+ channels, resulting in deteriorated K+ homeostasis and high K+ concentration in extracellular fluids.16 On the contrary, Na/ K-ATPase is involved in signaling during oxidative stress to alter cellular mechanisms and plays a key role in maintaining K+ homeostasis. Reactivation of Na/K-ATPase with released biomolecules thus paves a novel way to trigger cell defense reactions and restore cellular balance.17,18 Therefore, the biomimicking ECM that is designed to release biomolecules in response to ROS and K+ concentration and to reactivate Na/ K-ATPase represents a promising and persistent way to enhance ECM responsiveness to pathology and efficacy in converting diseased conditions to the normal state. Here, we present a novel strategy to mimic the ECM on an elastomer surface with electrospun micro-/nanofibers. First, we synthesize K+ and ROS dual-responsive tripolymer [poly(ethylene glycol)diacrylate, 1,2-ethanedithiol, and 4-nitrobenzo-18-crown-6-ether (PEGDA−EDT−BCAm)] containing ethylene glycol, thioether, and crown ether groups. Thioether and crown ether groups enable the tripolymer K+ and ROS dual-responsive, and ethylene glycol groups render the macromolecule biocompatible for tissue engineering.19 Then, acylated polycaprolactone (PCL-A)/PEGDA−EDT−BCAm fibers that encapsulate ascorbic acid-2 glucoside (AA-2G) are prepared through electrospinning. AA-2G is a stable form of ascorbic acid (AA)-glycoside under the conditions of high/low temperature or oxidation. Unlike AA, AA-2G is less susceptible to oxidation and easily encapsulated in micro-/nanofibers for a long time. After releasing from the fibers with responsiveness, AA-2G is readily adsorbed on the cell membrane and immediately metabolized by α-glucosidase on the membrane to further release activated AA. AA has proved to restrain ischemic reperfusion damage with its potent reductive effect.20 Therefore, AA-2G can not only remove excessive ROS in the extracellular microenvironment but also target Na/K-ATPase on the membrane to reactivate Na/K-ATPase for further triggering cellular defense reactions and achieving extracellular balance. Finally, high capability of the ECM to restore extracellular balance is demonstrated with model blood cells both in vitro and in vivo. This work establishes fundamentals

2. EXPERIMENTAL SECTION 2.1. Materials. Styrene-b-(ethylene-co-butylene)-b-styrene elastomer (SEBS, Kraton G 1652) was purchased from Shell Chemicals. PCL (Mn = 80 000 g/mol) and PEGDA (Mn = 1000 g/mol) were purchased from Sigma-Aldrich. AA-2G (Mw = 338.27 g/mol) was obtained from Tokyo Chemical Industry (Japan). 1,8-Diazabicyclo[5.4.0]-7-undecene (DBU, 98%) and 4-nitro-benzo-18-crown-6-ether (BCN with average Mn = 356 g/mol) were provided by Adamas Reagent Ltd. and TCI, respectively. Benzophenone (BP) and pentaerythritol tetra(3-mercapto propional) (PETMP) were purchased from Peking Ruichen Chemical (China). 2.2. Synthesis of PEGDA−EDT−BCAm. 4-Acrylamide-benzo18-crown-6 ether (BCAm) was synthesized from 4-nitro-benzo-18crown-6 ether (BCN) according to the previous works.19,21 In addition, the PEGDA−EDT polymer was synthesized by click reaction.22 Then, 1.0 g of purified PEGDA−EDT was dissolved in dimethyl sulfoxide under Ar gas for about 30 min, followed by introduction of BCAm at the equal molar ratio. The synthesis was performed in the presence of DBU at 60 °C for 15 h under the Ar atmosphere. The product was in the oil state after removing the solvent with a rotating evaporator and purified by column chromatography with a mixture of dichloromethane and methanol (6/1) as the eluent. Finally, the filtrate was collected and dried under vacuum for at least 2 days at 25 °C. All the monomers and synthesized products were analyzed in CDCl3 with a 400 MHz 1H NMR spectrometer (Bruker AV). 2.3. ROS and K+ Responsiveness of PEGDA−EDT−BCAm. The K+ responsiveness of BCAm was characterized by the turbidimetric method.23 BCAm was dispersed in deionized water to prepare a colloidal solution with a concentration of 5 × 10−3 M, and then 0.5, 1, and 2 molar equivalents of KCl solution (4 × 10−2 M) were added. After 20 min, the absorbency of all samples was determined by a TECAN absorbance reader (TECAN GENios, Austria) at 550 nm. The ROS responsiveness of PEGDA−EDT was characterized by the turbidimetric method. Seven hundred microliters of PEGDA−EDT solution (1.5 mg mL−1) was placed in a 48-well plate, followed by addition of 300 μL of H2O2 solution with concentrations of 0, 50, 100, and 150 mM. After 20 min, the optical density (OD) at 550 nm was determined with a microplate reader (Synergy H4, TECAN). PEGDA−EDT−BCAm (10 mg) was dissolved in 400 μL of D2O, and then 200 μL of H2O2/D2O (15 mM) solution and 200 μL of K+/ D2O (75 mM) were added to PEGDA−EDT−BCAm/D2O solution. After 12 h incubation at 37 °C, the responsiveness of PEGDA−EDT− BCAm to H2O2 (5 mM) and K+ (25 mM) was analyzed with an 1H NMR spectrometer. 2.4. Fabrication of the ECM with Electrospinning. Acylated PCL (PCL-A) was obtained through conjugation of acrylate onto hydroxyl groups of PCL.19,20 Then, a dry mixture of PCL-A, PEGDA−EDT−BCAm, and AA-2G was dissolved in mixed solvents of dimethylformamide and chloroform (40/60 wt %) at a concentration of 15 wt % (polymer/solution). Three compositions in the mixed solvents were prepared: PCL-A, PCL-A/PEGDA− EDT−BCAm (2/1 wt %), and PCL-A/PEGDA−EDT−BCAm/AA2G (6/3/1 wt %). To facilitate cross-linking reactions, 6 wt % PETMP and 1 wt % BP were added into the mixed solution. In addition, the mixed solution was transferred to a syringe for electrospinning. The micro-/nanofibers were fabricated on the SEBS substrate at room temperature with a solution feed rate of 0.8−1 mL/h and an applied voltage of 12.5−14.5 kV. The electrospinning was performed under UV irradiation. The distance between the collector and needles was 14−15 cm. The morphology of the biomimicking ECM was characterized with JEM1011 transmission electron microscopy (TEM, Japan) and Sirion100 field-emission scanning electron microscopy (SEM, USA). During electrospinning, the electrospun fibers were collected on B

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

20 μM Pi was used as the standard. The ATPase activity was calculated by eq 1

TEM copper grids for 20 s for TEM analysis. The sessile drop method was used to evaluate the surface hydrophilicity of the biomimicking ECM (∼300 μm thickness) with a contact angle goniometer (DSA, KRUSS GMBH, Germany) and about 3 μL of the water droplet. The biomimicking ECM was immersed in deionized water for a certain period from 3 to 24 h to examine the cross-linking reactions. X-ray photoelectron spectroscopy (XPS) (VG Scientific ESCA) equipped with an Al/K anode mono-X-ray source (hν = 1486.6 eV) was used to determine the surface composition of the ECM. Surface composition was also analyzed with a Bruker VERTEX 70 Fourier transform infrared (FTIR) spectrometer with an attenuated total reflection (ATR) unit (ATR crystal 45°) at a resolution of 4 cm−1 for 32 scans. The cytotoxicity of electrospun fibers to L929 murine fibroblast cells for 24−48 h was evaluated by CCK-8 assays. 2.5. ROS and K+ Dual Responsiveness of the ECM and AA2G Release. The biomimicking ECM (100 mg) was cut into the size of 1 cm × 1 cm and incubated in 11 mL of deionized water (a), phosphate-buffered saline (PBS) solution (b), 5 mM H2O2 solution (c), and 25 mM KCl solution (d). All the samples were stirred gently. After 8 h incubation, group (c) was added into 25 mM KCl solution (e). A solution (70 μL) was sampled at the certain time to measure the amount of released AA-2G with an instrument of TECAN (GENios, Austria) based on a standard calibration curve. The instrument was operated at 285 nm, and the content of AA-2G initially encapsulated in electrospun fibers was used to normalize the release profile. 2.6. Evaluation of Restoration Capability of the ECM in Vitro. 2.6.1. Determination of K+ and H2O2 Concentrations of Preserved RBCs. According to our previous work,24 400 μL of fresh concentrated RBCs from adult rabbits was diluted with 900 μL of NaCl solution and stored in the bags (400 mL) made of control SEBS and electrospun SEBS, respectively. The inner surface of the bags was covered with the biomimicking ECM. The bags were stored at 4 °C for 5 days. The K+-concentration of preserved RBCs was determined after 5day storage. The determination of K+ concentration is based on the fact that K+ reacted with sodium tetraphenylborate (STPB) in the presence of a protein precipitator to generate turbidity of suspension in alkaline media, and the turbidity is proportional to K + concentration. Erythrocyte (20 μL) was added into 180 μL of the protein precipitator and centrifuged at 3500 rpm for 5 min. A supernatant (50 μL), deionized water (negative control), and 0.4 mM potassium solution (positive control) were added into 200 μL of STPB solution and incubated for 5 min at 37 °C. The turbidity at 450 nm was determined by a TECAN GENios absorbance reader. The H2O2 concentration of stored RBCs was determined according to our previous method.24 Briefly, 50 μL of preserved RBCs was diluted with 1 mL of cold acetone to extract peroxides, followed by centrifugation (8000 rpm, 10 min, 4 °C) to obtain a peroxide supernatant. Then, 250 μL of the peroxide supernatant reacted with 25 μL of a titanium reagent for 5 min, and the precipitate of the peroxide−titanium complex was obtained with 50 μL of concentrated NH3·H2O. The complex was washed with acetone at least three times and dissolved in 250 μL of H2SO4 (2 M). The absorbency of the resulted solutions at 415 nm was determined by the TECAN GENios absorbance reader. 2.6.2. Na/K-ATPase Activity. The content of inorganic phosphorous (Pi) was measured to evaluate the ATPase activity based on the fact that the ATPase in the membranes decomposes ATP to ADP and Pi. Preserved RBCs (20 μL) were lysed hypotonically with 980 μL of deionized water, oscillated gently, and kept for 20 min. Then, 200 μL of lysed RBCs was cultured in a buffer for precise incubation for 10 min at 37 °C by adding the reaction terminator and centrifuged at 3500 rpm for 10 min to obtain the supernatant. Finally, a coloring developing reagent was dropped in the supernatant, and the phosphorus concentration was determined based on the OD at 636 nm by the TECAN absorbance reader. The control group referred to deionized water that was cultured in buffer and then added with a color developing reagent, and the blank group was only added with a coloring developing reagent without the buffer culture. The OD for

ATPaseactivity =

ODtest − ODcon × Cstd ODstd − ODbla

(1)

where ODtest is the OD of the test sample, ODstd is the OD of the standard, and ODcon and ODbla are the OD of the control sample and blank sample, respectively. 2.6.3. Malonaldehyde Content of Erythrocyte Membranes. Oxidation of preserved RBCs was characterized by the content of the lipid oxidative product, malonaldehyde (MDA), with a thiobarbituric acid method.25 Briefly, 400 μL of stored RBCs was lysed hypotonically with 12 mL of deionized water, oscillated gently, and kept for 20 min. The step for membrane separation from the supernatant by centrifugation (15 000 rpm, 20 min) at 4 °C was repeated three times to obtain the white/pale pellets of erythrocyte ghosts. Then, the hemoglobin-free ghosts were resuspended in 2 mL of PBS solution and preserved at 20 °C. The level of MDA in the membranes was determined with an assay kit of Jian Cheng (Ultramicro MDA test kit, China). Meanwhile, 150 μL of membrane suspension in deionized water was used to measure the concentration of membrane protein through the bicinchoninic acid (BCA) method. The amount of MDA was expressed as the histogram of MDA per nanomole of the membrane protein. 2.6.4. Hemolysis Assay. Preserved RBCs (50 μL) were dispersed in 1 mL of NaCl solution with the concentrations of 0.9, 0.7, 0.5, 0.3, and 0.1%. Fresh RBCs (0.1 mL) were added to 2 mL of deionized water and saline (0.9%) to set positive and negative controls, respectively. The RBCs were incubated for 2 h at 37 °C and centrifuged (3000 rpm, 3 min) to obtain the supernatant. Then, 70 μL of the supernatant was dropped into 96-well plates to determine the OD at 541 nm by the TECAN absorbance reader. In addition, the hemolysis ratio (HR) was determined based on eq 2 HR(%) =

ODtest − ODneg ODpos − ODneg

× 100 (2)

where ODtest, ODpos, and ODneg are the absorbance of the test sample, positive control, and negative control, respectively. 2.7. Evaluation of Restoration Capability of the ECM in Vivo. Six healthy young rabbits (six-month old) were divided into two groups: A and B. Blood (2 mL) was obtained from the ear artery of each rabbit by puncture and collected in a medical blood collection tube (SANLI Company) with 0.2 mL of sodium citrate solution (3.8 wt %) at room temperature. The RBCs were separated from whole blood by centrifugation (1000 rpm for 15 min), and upper plasma and leucocyte were sequentially removed by a sterilized syringe. The remaining RBCs were washed with three volumes of PBS solution and centrifuged at 1000 rpm for 5 min. The RBCs were diluted with sterilized KCl/PBS solution (50 mM) at a ratio of 1/5. PCL-A/ PEGDA−EDT− BCAm/AA-2G meshes (100 mg) were sterilized with 75% alcohol and UV irradiation. The RBCs from group A were treated with the sterilized meshes by immersion of meshes in the RBC suspension. The RBCs from group B remained untreated. All blood was preserved in the dark at 4 °C for 2 h and then moved to an incubator at 38 °C for 20 min. Transfusion was carried out according to the report by Hod et al.26 During transfusion, 10 mL of RBCs was injected to the corresponding rabbit through the ear vein with a speed of 1 mL/min. K+ and the serum amyloid protein (SAA) concentrations in the blood were measured at 0.5, 1, and 1.5 h. SAA was measured by the rabbit serum amyloid A (SAA) enzymelinked immunosorbent (immunoadsorbent) assay kit. 2.8. Statistical Analysis. The K+ concentration, H2O2 concentration, HR, MDA contents, and SAA content are presented as means ± standard deviation for the indicated number of erythrocytes. Origin software was used for statistical analysis. In addition, post hoc analysis was employed with the test of Bonferroni’s multiple comparison when it was appropriate. Differences were considered statistically significant at P ≤ 0.05. C

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

3. RESULTS AND DISCUSSION Biomaterials’ implantation always causes tissue or organ damage during the surgical procedure. The injury induces an inflammatory response to biomaterials with increasing ROS in the extracellular environment.3,4 Na/K-ATPase is the target for oxidation and highly susceptible to oxidative damage directly.14 In addition, the oxidation of Na/K-ATPase directly leads to excessive cation leak and a rapid increment of extracellular potassium ion (K+) concentration. In return, the K+ efflux from the cell activates pyrin domain-containing 3 (NLRP3) inflammasome, which induces ROS and exaggerates oxidative stress.27 As a result, the vicious circle between overproduction of ROS and ion imbalance leads to excessive ROS and K+ in the extracellular environment and inactivity of Na/K-ATPase. Combination of K+ and ROS responsiveness is thus expected to enhance the sensitivity of the ECM to pathological changes substantially. Moreover, because Na/K-ATPase is involved in signaling during oxidative stress to alter cellular mechanisms and activate the cell defense system,17,18 delivery of antioxidants to the membrane and reactivation of Na/KATPase pave a novel way to restore extracellular ion and ROS balance by the biomimicking ECM (Figure 1A). The ECM is

Figure 2. Pathway for the synthesis of PEGDA−EDT−BCAm (A) and 1H NMR spectra of BCAm and the synthesized PEGDA−EDT− BCAm (B). 1H NMR spectra confirm successful synthesis of a triblock copolymer.

of the overdose of EDT, thiol groups remain in the chain terminal and provide active sites for the subsequent reaction. Then, BCAm is prepared by the reduction of the nitro group (−NO2) in the benzene ring of 4-nitro-benzo-18-crown-6 to amidogen (−NH2) with Pd/C catalysts, followed by acylation with acryloyl chloride (steps 2−3 in Figure 2A). Finally, the tripolymer of PEGDA−EDT−BCAm is obtained through the thiol−ene reaction between PEGDA−EDT and BCAm at an equal molar ratio (step 4 in Figure 2A). The 1H NMR spectra of BCAm and PEGDA−EDT−BCAm are shown in Figure 2B. Typical signals of protons connected with the benzene ring (7.45, 6.91, 6.83 ppm) and that in the acryloyl group (5.8−6.6 ppm) are detectable in the spectrum of BCAm.28 Compared with the spectrum of BCAm, the signals for protons in the acryloyl group disappeared, but the chemical shifts of protons connected with the benzene ring (7.45, 6.91, and 6.83 ppm) and new signals for thioether groups are observable in the spectrum of PEGDA−EDT−BCAm, confirming successful synthesis of a triblock copolymer. Based on the GPC trace of PEGDA−EDT−BCAm in THF (Supporting Information, Figure S2), the average molecular weight (Mn) of PEGDA− EDT−BCAm is 3.253 × 104, with the molecular weight distribution of 1.62 (Supporting Information, Figure S2). PCL acylation is carried out through conjugation of acryloyl chloride onto the hydroxyl groups of PCL at the terminal (Figure 3A). Comparison of 1H NMR spectra of PCL and PCL-A confirms the successful acylation (Figure 3B). Based on the relative peak area at 5.8−6.6 ppm (acryl protons) and peak area at 1.0−1.5 ppm (methylene protons),19 the degree of acylation is determined to be 80−95%. Acylation entitles PCL with unsaturated groups, which facilitate reactions between PCL-A and PEGDA−EDT−BCAm during electrospinning.24 3.2. ECM Preparation and Surface Characterization. PCL-A, PEGDA−EDT−BCAm, AA-2G, BP, and PETMP are dissolved in the solution of dimethylformamide and chloroform. Then, the micro-/nanofibers are fabricated on the SEBS

Figure 1. Biomimicking dual-responsive ECM restoring extracellular balance through the Na/K-ATPase pathway. (A) Biomimicking ECM with K+ and ROS dual responsiveness maintains K+ and ROS balance. (B) Schematic illustration of ECM fabrication and interaction between the ECM and cells. The vicious circle between ROS overproduction and ion imbalance causes damage to the cell membrane and Na/K-ATPase, and the biomimicking ECM with K+ and ROS dual responsiveness breaks the vicious circle by removing the excessive toxicity and restoring extracellular K+ and ROS homeostasis through the Na/K-ATPase pathway.

composed of electrospun micro-/nanofibers of the K+ and ROS-responsive triblock copolymer (PEGDA−EDT−BCAm) and PCL that capsulate AA-2G. The ECM releases AA-2G in response to excessive K+ and ROS and restores the extracellular balance through the Na/K-ATPase pathway (Figure 1B). 3.1. Synthesis of PEGDA−EDT−BCAm and PCL-A. The PEGDA−EDT−BCAm triblock copolymer contains ethylene glycol, thioether, and crown ether groups such that it exhibits high biocompatibility and K+ and ROS dual responsiveness.21−23 PEGDA−EDT−BCAm is synthesized by two-step click reactions. First, the PEGDA−EDT copolymer is prepared through a thiol−ene reaction between PEGDA and EDT (step 1 in Figure 2A, Supporting Information, Figure S1). PEGDA− EDT possesses ROS sensitivity because the backbone of polymer chains contains oxidizable thioether groups.24 Because D

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

chemical reactions between PCL-A and PEGDA−EDT− BCAm occur in the electrospinning process (Supporting Information, Figure S3) so that the stability of electrospun meshes on SEBS is enhanced substantially. Figure 4d,e shows the SEM images of PCL-A/PEGDA−EDT−BCAm fibers with and without cross-linking reactions after 4 day immersion in deionized water, respectively. The former with coupling reactions exhibits swelled fibers (Figure 4d), but the latter without cross-linking reactions shows many pores in fibers, indicating the dissolution of the PEGDA−EDT−BCAm component from fibers (Figure 4e). FTIR spectra further confirm the stable meshes prepared through reactive electrospinning (Figure 4f). In comparison with the disappearance of a peak at 1607 cm−1 (−NH stretching vibration of PEGDA− EDT−BCAm) in the spectrum of noncross-linked fibers [Figure 4f(III)], the peak remains in the spectrum of crosslinked fibers after immersion in water for 4 days [Figure 4f(II)]. Because of the difference in the molecular structure and immiscible blends ofPEGDA−EDT−BCAm and PCL-A, the low viscous PEGDA−EDT−BCAm chains tend to reside in the sheath part and hydrophobic PCL-A in the core during electrospinning,24,29 resulting in the core−shell structure of fibers and the hydrophilic surface of fibers (insets of Figure 4b,c). TEM images of the PCL-A/PEGDA−EDT−BCAm fiber exhibit a core−shell structure obviously, confirming the occurrence of phase separation during electrospinning. XPS spectra show that PEGDA−EDT−BCAm tends to cover the fiber surface (Figure 4h). The core−sheath structure of the microfiber facilitates encapsulating antioxidants, AA-2G, without affecting fiber hydrophilicity. The cytotoxicity of ECM is evaluated with the L929 murine fibroblast cell as the model. The cell viability remains higher than 80% after incubating

Figure 3. (A) PCL acylation pathway and (B) 1H NMR spectra of PCL (a) and PCL-A (b). The degree of acylation is determined to be 80−95% based on 1H NMR spectra.

substrate by reactive electrospinning to mimic the ECM. In addition, the electrospun fibers of PCL-A and PCL-A/ PEGDA−EDT−BCAm (50/50 wt %) without AA-2G and BP are prepared for comparison, respectively. The water contact angle (WCA) of PCL-A microfibers is 121°, showing the hydrophobic surface of PCL-A fibers (Figure 4a). Electrospinning of the two components of PEGDA−EDT− BCAm and PCL-A makes the microfibers uniform without beads (Figure 4b). In addition, the fiber surface becomes superhydrophilic as the value of WCA is about 0°. The fiber morphology and surface hydrophilicity are slightly influenced in the presence of AA-2G (Figure 4c). The UV-induced

Figure 4. Structure and surface characterization of the biomimicking ECM. SEM images of the ECM composed of (a) PCL-A, (b) PCL-A/ PEGDA−EDT−BCAm, (c) PCL-A/PEGDA−EDT−BCAm/AA-2G electrospun fibers, (d,e) cross-linked and noncross-linked PCL-A/PEGDA− EDT−BCAm fibers soaked in water for 4 days, respectively; (f) FTIR spectra of the PCL-A fiber (fI), cross-linked PCL-A/PEGDA−EDT−BCAm fiber (fII) and noncross-linked PCL-A/PEGDA−EDT−BCAm fiber (fIII) after 4-day immersion in deionized water. (g) TEM image of PCL-A/ PEGDA−EDT−BCAm/AA-2G electrospun fibers; (h) XPS of PCL-A/PEGDA−EDT−BCAm/AA-2G electrospun fibers; (i) cell toxicity evaluation of the ECM; the insets in (a−c) are photographs of WCA for corresponding ECM. E

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

5B). The responsiveness of PEGDA−EDT−BCAm to K+ and H2O2 is further analyzed with 1H NMR spectra (Figure 5C). Compared with the spectrum of PEGDA−EDT−BCAm [Figure 5C(a)], the K+/BCAm complex induces a small upfield shift of protons on the crown ether ring [Figure 5C(c)], which is in agreement with the report of Liu et al.33 Hydrophilic sulfoxide and sulfone groups (3.2−3.4 ppm) appeared after oxidation by H2O2 [Figure 5C(b)]. Both the K+/BCAm complex and formation of sulfoxide and sulfone groups render PEGDA−EDT−BCAm hydrophilic to absorb water easily.22,23 The K+ and ROS dual responsiveness of PEGDA−EDT− BCAm enable the fast responsiveness of the ECM to pathological changes to release AA-2G. The ECM responds to K+ at the concentration of 50 mM in less than 5 min (Supporting Information, Figure S7). The dependence of AA2G release on H2O2 and K+ concentration is shown in Figure 5D. The release of AA-2G from the ECM with the 5 mM H2O2 (curve c) or 25 mM K+ solution (curve d) is much faster than that in the deionized water (curve a) and PBS (b) solution. When exposed to H2O2 and K+, PEGDA−EDT−BCAm chains become water-soluble to render the fiber surface hydrophilic.22,23 As a consequence, the hydrophilic fibers tend to adsorb water molecules and become swelled, resulting in the accelerated release of AA-2G.24 Moreover, when the electrospun fibers are first immersed in 5 mM H2O2 for 8 h and then in the mixed solution of 5 mM H2O2 and 25 mM K+, the release becomes fast and the total amount of release is highest (curve e). This demonstrates that the dual responsiveness of electrospun fibers increases the sensitivity of the ECM to excessive ROS and K+. Compared with AA, AA-2G is less susceptible to oxidation and can be easily stored in micro-/ nanofibers for a long time. After releasing from the fibers with dual responsiveness, AA-2G is readily adsorbed on the cell membrane and immediately metabolized by α-glucosidase on the membrane to further generate activated AA. Activated AA is confirmed to possess its potent reduction in inhibiting ischemic-reperfusion injury.20 The generated AA around the membrane removes excessive ROS and protects Na/K-ATPase from oxidation. Therefore, AA-2G can target the cell membrane and reactivate Na/K-ATPase to trigger cellular defense reactions and restore K+ and ROS balance.34 3.4. Restoring Extracellular Balance with the ECM. The capability of the ECM to restore extracellular balance is evaluated in vitro and in vivo. RBCs are selected as the model because they are anuclear and ideal for investigation of interactions between the ECM and cells.35 In addition, RBCs are the most abundant cells in the blood, which play key roles in oxygen/nutrient transportation, blood-based illness diagnosis and detection, and immuneprotection.36,37 To simulate the conditions of excessive ROS and ion imbalance, 400 μL of RBCs in 0.9% NaCl solution (60 Vol %) is stored in the bags at 4 °C for 5 days. These bags are made of control SEBS and electrospun SEBS film, and the inner surface of electrospun SEBS is covered with the ECM. The photograph of RBCs in bags is shown in Figure S8 (Supporting Information, Figure S8). To check the ECM efficiency clearly, no commercial additive is added to the bags. During RBC storage, the reduction of lactate and drop in pH lead to the deficiency of antioxidant enzymes, which causes the ROS accumulation and loss of cation gradients across the membrane.38 After storage for 5 days, the K+ and H2O2 concentrations in the bags of control SEBS and electrospun

with ECM, indicating high cell compatibility of the ECM. Moreover, the ECM surface resists protein adsorption of bovine serum albumin and fibrinogen as well as platelet adhesion (Supporting Information, Figure S4). In addition, compared to control SEBS, the ECM substantially elongates blood clotting time (Supporting Information, Figure S5), demonstrating high blood compatibility. To further confirm the biocompatibility of the ECM, subcutaneous implantation of the ECM to mice is performed (Supporting Information, Figure S6). The interleukin factor, IL-6 level, in the blood of mice after SEBS implantation is much higher than that of normal mice. In contrast, the IL-6 level in the blood of ECMimplanted mice is almost similar to that in normal mice, indicating that the biomimicking ECM exhibits high biocompatibility. The ECM provides a dynamic microenvironment to constantly signal cells,13 and high biocompatibility renders the ECM suitable for cell manipulation both in vitro and in vivo. 3.3. Responsiveness of ECM and Targeted AA-2G Delivery. Responsiveness to adventitious stimuli is the fundamental feature for the ECM to manipulate extracellular conditions.11,30−32 The K+ responsiveness of BCAm is analyzed by the turbidimetric method. BCAm colloidal solution is added to KCl solution at the K+/BCAm molar ratios of 0, 0.5, 1, and 2. The complexion between BCAm and K+ (insets of Figure 5A) makes the solution tunable with

Figure 5. Responsiveness of PEGDA−EDT−BCAm and ECM to K+ and H2O2. (A) Responsiveness of BCAm to K+; (B) responsiveness of PEGDA−EDT to H2O2; (C) 1H NMR spectra of PEGDA−EDT− BCAm (a) without treatment, (b) after reaction with 5 mM H2O2, and (c) after treatment with 25 mM K+, respectively; (D) release profiles of AA-2G from the ECM in (a) deionized water, (b) PBS, (c) 5 mM H2O2, (d) 25 mM K+, and (e) 5 mM H2O2 + 25 mM K+ solution. Dual responsiveness of electrospun fibers increases the sensitivity of the ECM to excessive ROS and K+.

varied turbidity. The OD attenuates with enhanced K+ concentration, confirming the sensitivity of BCAm to K+ concentration (Figure 5A). Similarly, the responsiveness of PEGDA−EDT to ROS is analyzed by feeding PEGDA−EDT solution to H2O2 solution. Because the hydrophobic thioether groups are converted into hydrophilic sulfone and sulfoxide groups with oxidation (insets of Figure 5B), PEGDA−EDT becomes soluble with increasing H2O2 concentration (Figure F

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

Figure 6. Restoration of extracellular balance with the biomimicking ECM in vitro. (A) K+ and H2O2 concentration in blood bags; (B) activity of Na/K-ATPase; and (C) MDA content of preserved RBCs in different blood bags; (D) dependence of hemolysis of preserved RBCs on varied NaCl concentration. The inner surface of SEBS bags is coated with electrospun fibers whose component is listed in the figures clearly. The biomimicking ECM exhibits high capability to restore extracellular balance through the Na/K-ATPase pathway.

Figure 7. Restoration capability of the biomimicking ECM in vivo. (A) Schematic illustration of transfusion of rabbits with RBCs containing high content of K+ originally. One group is transfused with RBCs treated by the ECM and the other with RBCs without treatment by ECM; (B) K+ concentration in the blood of rabbits after transfusion; and (C) concentration of SAA in the blood of rabbits after transfusion.

PCL-A increase significantly compared with fresh RBCs. On the contrary, the K+ and H2O2 concentrations in electrospun PCL-A/PEGDA−EDT−BCAm and PCL-A/PEGDA−EDT− BCAm/AA-2G bags remain low (Figure 6A). The K+ and H2O2 levels in the latter are 6.18 and 0.15 mM, respectively, which are close to the normal levels of fresh RBCs (∼5 mM and 0.1 mM, respectively). In addition, RBCs in the bags of PCL-A/PEGDA−EDT−BCAm and PCL-A/PEGDA−EDT−

BCAm/AA-2G possess much higher activity of Na/K-ATPase than that in the control SEBS bags and electrospun PCL-A bags (Figure 6B). The ATPase activity in varied K + concentrations shows that the Na/K-ATPase activity in the 25 mM, 15 mM K+ solution is significantly lower than that in PBS (Supporting Information, Figure S9). Because high ROS and K+ concentration in extracellular fluids renders Na/KATPase inactive, high activity of Na/K-ATPase confirms the G

DOI: 10.1021/acsami.9b05420 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces capability of the ECM in removing toxic substances and reactivating Na/K-ATPase. Furthermore, high activity of Na/ K-ATPase facilitates triggering cellular defense reactions to attenuate oxidative stress and restore extracellular balance.18 Oxidative stress may cause lipid oxidation in the membrane and consequent hemolysis of preserved RBCs. The content of the oxidative product of the membrane lipid, MDA, is shown in Figure 6C. The lowest content of MDA is observed for the RBCs in the bag of electrospun PCL-A/PEGDA−EDT− BCAm/AA-2G, which is close to the content of fresh RBCs (5 nmol/mg protein). The HR of RBCs in different bags on NaCl concentration (osmotic pressure) is shown in Figure 6D. The hemolysis of RBCs decreases from PCL-A, SEBS, PCL-A/ PEGDA−EDT−BCAm to PCL-A/PEGDA−EDT−BCAm/ AA-2G bag at each saline concentration, indicating that the ECM restores the ROS and ion homeostasis to maintain cell normal function. 39 Moreover, the ECM capability in maintaining cell normal function is demonstrated by the high ratio of normal shapes of preserved RBCs (Supporting Information, Figure S10) and low degree of membrane oxidation of stored RBCs (Supporting Information, Figure S11) in the bag of PCL-A/PEGDA−EDT−BCAm/AA-2G. The restoration capability of the ECM is further evaluated in rabbits with RBC transfusion. Fresh RBCs (2 mL) from the rabbit are collected in medical blood collection tubes and diluted with 50 mM KCl/PBS solution at a ratio of 1/5. RBC suspension in KCl/PBS solution is divided into two groups. Then, RBC suspension in one group is pretreated with PCLA/PEGDA−EDT−BCAm/AA-2G fibers by immersion of the fibers in the suspension and the other is without any treatment. After storage in the dark at 4 °C for 2 h and subsequent incubation at 38 °C for 20 min, RBCs are transfused to the corresponding rabbit at a speed of 1 mL/min (Figure 7A). The blood is sampled from the ear artery to measure K+ concentration and the serum amyloid protein (SAA) at 0.5, 1, and 1.5 h. Addition of KCl to RBC suspension causes the high K+ concentration in the RBC suspension (hyperkalemia), which can further induce ROS generation and inflammation in vivo, as well as deactivation of Na/K-ATPase. SAA is an acute phase protein that rapidly increases by about 1000 times within 4−6 h after the body is infected. Thus, it is a sensitive indicator reflecting the body’s inflammation.40 After transfusion with nontreated RBCs (control), high K+ concentration (Figure 7B) and SAA concentration (Figure 7C) in the blood are detected. In contrast, both K+ and SAA concentrations in the blood remain nearly as normal as fresh RBCs after transfusion with treated RBCs. The above results show that the treated RBCs do not induce high K+ concentration and inflammation after transfusion. Therefore, the biomimicking ECM shows high capability to restore extracellular balance through the Na/KATPase pathway both in vitro and in vivo. This method is versatile and effective, which provides basic principles to prepare self-adaptive implant biomaterials for next generation.

extracellular balance. We demonstrated that the synthesized tripolymer of PEGDA−EDT−BCAm exhibited ROS and K+ responsiveness; PCL-A/PEGDA−EDT−BCAm/AA-2G micro-/nanofibers was fabricated through reactive electrospinning and the fibers released AA-2G in response to the excessive K+ and ROS in an extracellular microenvironment; and the delivery of AA-2G to the cell membrane and generation of AA by α-glucosidase not only removed the toxic substances around cells but also reactivated Na/KATPase in the cell membrane. Therefore, the biomimicking ECM exhibited high capability to reduce oxidative stress and restored extracellular homeostasis through the Na/K-ATPase pathway both in vitro and in vivo. This work provides basic principles to design an implant that mimics the ECM to avoid pathogenicity and improve implant integrity and longevity in vivo.

4. CONCLUSIONS We developed a new strategy for an implant that mimics the physiological ECM to modulate the host response and improve implant integrity and longevity. This strategy was based on the fact that ROS and K+ dual responsiveness enhanced the sensitivity of the ECM to pathological changes and delivery of AA-2G from the ECM to cell membrane favored reactivating Na/K-ATPase to trigger cellular defense reactions and restore

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b05420. 1 H NMR spectra of PEGDA, EDT, and PEGDA−EDT; GPC trace of PEGDA−EDT−BCAm in THF; coupling reactions during electrospinning; protein adsorption and platelet adhesion; blood clotting index on the SEBS and electrospun fibers; IL-6 level on the mice blood after implantation; K+ adsorption by the PCL-A/PEGDA− EDT−BCAm fiber; RBC preservation in bags at 4 °C for 5 days; Na/K-ATPase activity on varied K + concentrations; SEM images of preserved RBCs in the bags; and CLSM images of preserved RBCs in SEBS bags, electrospun PCL-A bags, PCL-A/PEGDA−EDT− BCAm bags, and PCL-A/PEGDA−EDT−BCAm/AA2G bags (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Y.F.). *E-mail: [email protected]. Phone: +86 431 85262388. Fax: +86 431 85262126 (Q.S.). ORCID

Xingkun Luan: 0000-0001-7828-7329 Zhifang Ma: 0000-0001-8297-0876 Qiang Shi: 0000-0003-4431-4434 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was financially supported by the National Key Research and Development Program of China (2016YFC1100402), the National Natural Science Foundation of China (51573186, 21807097), and the National Natural Science Foundation of Jilin Province (20180101178JC, 20190701030GH). REFERENCES

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