PEI Polyelectrolytes Hydrogel

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A Novel Balanced Charged Alginate/PEI Polyelectrolytes Hydrogel that Resists Foreign-Body Reaction Jiamin Zhang, Yingnan Zhu, Jiayin Song, Jing Yang, Chao Pan, Tong Xu, and Lei Zhang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b17670 • Publication Date (Web): 02 Feb 2018 Downloaded from http://pubs.acs.org on February 5, 2018

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ACS Applied Materials & Interfaces

A Novel Balanced Charged Alginate/PEI Polyelectrolytes Hydrogel that Resists Foreign-Body Reaction Jiamin Zhang1,2,3, Yingnan Zhu1,2,3, Jiayin Song1,2,3, Jing Yang1,2,3, Chao Pan1,2,3, Tong Xu1,2,3, Lei Zhang1,2,3,* 1

Department of Biochemical Engineering, School of Chemical Engineering and Technology,

Tianjin University, Tianjin 300072, P.R. China  2

Key Laboratory of Systems Bioengineering (Ministry of Education), Tianjin University, Tianjin

300072, P.R. China  3

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin

University, Tianjin, 300072, P.R. China E-mail: [email protected]

KEYWORDS： balanced charged hydrogel; foreign body reaction; antifouling; polyelectrolytes; alginate

ABSTRACT: Foreign-body reaction (FBR) has been a long-term obstacle for implantable biomedical devices and materials, especially those require mass/signal transport between the implants and the body. However, currently very limited biomaterials can mitigate FBR. In this work, we develop a balanced charged polyelectrolytes hydrogel that can efficiently resist FBR and collagenous capsule formation in a mouse model. Using this new strategy, we can easily tune the antifouling properties of the polyelectrolytes hydrogels by changing the ratio of negatively

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charged alginate and positively charged poly (ethylene imine) (PEI). We find that at the optimum ratio where the net charge of hydrogel is neutral, the adhesion of proteins, cells, bacteria and fresh blood on its surface can be significantly inhibited, indicating its excellent antifouling properties. In vivo studies show that after implanted subcutaneously, this balanced charged hydrogel can prevent the capsule formation for at least 3 months. Furthermore, immunofluorescent staining results indicate that this balanced charged hydrogel elicits negligible inflammation, significantly reduces macrophage migration to the tissue-implant interface. This flexible and versatile approach holds a great promise for designing a wide spread of new antifouling hydrogels and using as immunoisolation materials for biomedical applications.

Introduction Implantable devices or biomaterials play a key role in biomedical applications including controlled drug delivery, tissue engineering, medical sensors, etc.

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However, foreign-body

reaction (FBR) always poses an inherently obstacle to their successful applications.4,5 It has been known that the immune system will recognize the implant as a foreign object, and ultimately wall it off with a dense avascular collagenous capsule a couple of weeks after the implantation. This fibrous capsule is an impermeable barrier that blocks mass/signal transport and isolates the implants from body, consequently causes implantation failure and increases the risk of requiring secondary or even more surgeries. 6 Recently, it has been found that immune-recognition and FBR to implants were triggered by the nonspecific protein adsorption on their surfaces. When devices or biomaterials enter the body, they are immediately coated with various types of proteins. Then these protein-labeled implants


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are firstly attacked by neutrophils, which also recruit macrophages to digest these implants. But when the uptake or digestion fails, the macrophage cells would accumulate and fuse to larger foreign body giant cells (FBGCs). However, FBGCs are still too small to internalize the implants, so they secrete cytokines that recruit fibroblast cells to encapsulate the implants by a dense collagenous capsule.4,7 Therefore, the key to resist FBR is to eliminate surface protein adsorption at the first place. The development of antifouling materials that can efficiently prevent the undesirable proteins adhesion have attracted increasing attentions. Recently, it has been found that zwitterionic materials exhibited strong ability to resist the adhesion of proteins, cells and bacteria.5 This is because each zwitterionic unit is consisted of one positive charge and one negative charge which are overall neutrally charged, thus it can strongly bind to water molecules and efficiently eliminate the interaction with biomolecules.8,9 Especially, polycarboxybetaine (PCB) is a very important one and possesses several unique properties. Jiang et al. demonstrated that the PCB coated surfaces could achieve undetectable protein adsorption from complex media.9-11 More importantly, they demonstrated that after subcutaneously implanted into mice, PCB hydrogels could inhibit FBR and avoid capsule formation for at least 3 months.12 Motivated by the excellent antifouling properties of zwitterionic materials, researchers have also attempted to develop alternative ways to mimic zwitterionic surfaces. Chen et al. demonstrated that antifouling surfaces could also be constructed from mixed self-assembled monolayers (SAMs) with balanced counter-charged (monovalent) groups from different molecules.13,14 This result suggested that the balanced charge formed by either zwitterioinc or mixed charged groups was the key to the antifouling properties of these surfaces. And it also


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opened a new opportunity to develop various antifouling surfaces with a wide range of equal valence molecules. The successful development of balanced charged 2-dimension antifouling surface inspired us to further explore 3-dimension (3-D) structured antifouling materials. In this work, we hypothesize that the assembly of oppositely charged macromolecules (e.g. polyelectrolytes) may be used to develop antifouling bulk hydrogels. Alginate was used as the negatively charged polyelectrolyte and poly (ethylene imine) (PEI) was used as the positively charged polyelectrolyte. A series of differently charged bulk hydrogels could be easily prepared by tuning the ratios of alginate and PEI. It was found that the balanced charged hydrogel could efficiently prevent the biofouling adhesion. Most importantly, it could also avoid the immune response and capsule formation in a subcutaneous implantation mice model. Experimental section Materials. Sodium alginate was purchased from Sigma-Aldrich (USA). Branched poly (ethylene imine) (PEI, Mw=70 kDa) was obtained from Aladdin® Company (Shanghai, China). Calcium chloride (CaCl2) was purchased from Macklin Biochemical Technology co., Ltd (Shanghai, China). Hematoxylin & eosin (H&E) kit, Masson’s trichrome kit and fluorescein isothiocyanatelabeled bovine serum albumin (BSA-FITC) were purchased from Solarbio Science & Technology Co., Ltd (Beijing, China). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S) and Live/Dead kits were obtained from Invitrogen (Carlsbad, USA). Other chemical regents were purchased from Tianjin Chemical Reagent Company (Tianjin, China).


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Preparation of Alg/PEI polyelectrolyte hydrogels. All the alginate was purified before used in this work.15 Alg/PEI hydrogels were prepared by mixing negatively charged alginate and positively charged PEI with different ratios. Briefly, PEI solution (1%, w/v) was slowly added to alginate solution (1%, w/v) with stirring, and then the mixture further crosslinked by CaCl2 solution (10%, w/v). Five different hydrogels were made by tuning the alginate: PEI ratios, including 1:0, 1:0.025, 1:0.045, 1:0.06 and 1:0.1 (w/w). And the proportion of alginate: Ca2+ was 2:3 (w/w). Then the zeta potential of five Alg/PEI hydrogels was measured using Zetasizer Nano ZS (Malvern, UK). Water content tests. Five formulations of Alg/PEI hydrogels were obtained as described above and punched into 1 cm-diameter disks before tests. The hydrogel samples were rinsed with the deionized water for 5 times and weighted as wet mass. Then the samples were dehydrated at 60 °C in vacuo. After 3 days, the dried hydrogel samples were weighted again. 5 samples of each hydrogel formulation were measured and calculated with the equation (1): Water content (%) = (mw -md)/mw × 100

(1)

Where mw and md were the mass of the wet hydrogel and the dry hydrogel respectively. Mechanical property tests. The compressive modulus of all hydrogels was measured with a microcomputer control mechanical tester (WDW-5, Beijing, China). Five 1 cm-diameter hydrogel disks of each formulation (0.5 cm thickness) were compressed at a rate of 1 mm/min. The compressive modulus was calculated from 10% to 25% strain. In vitro cytotoxicity and endotoxin tests. MTT tests were used to evaluate the cytotoxicity of the hydrogel samples. To extract any soluble impurities, the samples were immersed and


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incubated into the DMEM medium supplemented with 10% FBS and 1% P/S for 24 h. At the same time, the NIH-3T3 fibroblasts were seeded into a 48-well plate at concentration of 5×105 cells/mL and cultrued for 24 h at 37 °C. Then, the culture medium was discarded from the cells and 500 µL extracting medium was then added. After 2 days, MTT reagents were added and the absorbance of the final purple suspensions was measured with the microplate reader (Tecan infinite 200 PRO, Switzerland) at 490 nm. Limulus Amebocyte Lysate (LAL) endotoxin assay kit ((Limulus Amebocyte Lysate Company, China) was used to tested the endotoxin content of hydrogels. All the hydrogel samples showed the endotoxin level was less than 0.06 EU/mL which is the detection line of the endotoxin. Protein adsorption tests. In these tests, FITC labeled BSA was used as the model protein. Hydrogels samples of each formulation were washed with 0.9% NaCl solution for several times before tests. Then, they were placed into a 48-well plate and incubated with 0.5 mg/mL BSAFITC solutions for 30 min away from the light. After washing for 3 times with 0.9% NaCl solution, the samples were observed using an inverted fluorescence microscope (Nikon Eclipse Ti-S, Japan). And the relative fluorescent intensity was normalized to the area of observed hydrogel using Image J software. Five samples of each hydrogel formulation were measured. Cell adhesion and cell viability. The 5 mm-diameter hydrogel disks were placed into a 48-well plate and irradiated with UV light for 1 h. 100 µL NIH-3T3 cell suspensions (5×105 cells/mL) were added into the well and incubated at 37 °C under 5% CO2 for 72 h. The hydrogels were observed and photographed with an invert microscope. Five areas of each hydrogel surface were photographed and counted the number of adherent cells from each image.


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The viability of cells on the hydrogel surfaces was evaluated using Live/Dead kit. After 72 h of culture, the cell-hydrogel disks were gently rinsed twice with 0.9% NaCl solutions. Then the cells were stained by the calcium-AM (green, live) and ethidium homodimer (red, dead) mixture solutions at 37 °C for 30 min and observed by an inverted fluorescence microscope. The number of live and dead cells were counted to calculate the viability. Bacterial adsorption tests. The bacterial specie, gram-negative bacteria (Escherichia coli) was used in this test. 1 cm-diameter hydrogel disks were sterilized by UV irradiation for 1 h before test. Then the hydrogel disks were incubated in broth with the bacterial suspension (1×107 cells/mL) at 37 °C under orbital stirring for 2 h. Then, the hydrogels were aseptically placed into a new 24-well plate and rinsed 3 times with 0.9% NaCl solution to remove any free bacteria. The last washing fluid (100 µL) was spread on LB Agar plates. After 24 h incubation, the colonyforming units (C.F.U.) were calculated. Five different samples of each hydrogel were measured. Hemocompatibility tests. The hemocompatibility of each hydrogel formulations was evaluated with blood cells adhesion tests and hemolysis assays. The 1 cm-diameter hydrogel disks were incubated in whole blood at 37 ℃. After 30 mins, the samples were gently rinsed twice with 0.9% NaCl solutions and observed. Five different samples of each type of hydrogel were measured. Hemolysis ratio (HR) measurement was used to investigate the hemolysis effect of the hydrogels.16 Briefly, fresh blood was five times diluted with 0.9% NaCl solution and then coincubated with hydrogel samples at 37 °C for 30 min. The positive control was deionized water diluted fresh blood, and the negative control was the 0.9% NaCl solutions diluted fresh blood.


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After centrifuged at 1000 rpm for 10 min, the supernatants of the samples were collected and the absorbance was measured with the microplate reader at 541 nm.

To calculate the HR of all samples, equation (2) was used: HR = (AS-AN) / (AP-AN)

(2)

Where AS, AP and AN were the absorbance of the samples, positive control and negative control, respectively.

Hydrogel samples implantation. All animal procedures in this work were followed the regulations of the committee of the Laboratory Animal Science Department of Tianjin Medical University. BALB/c female mice (8 weeks, 25g) were provided by the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). Hydrogel samples with different charge (Alg/PEI0, Alg/PEI0.045, Alg/PEI0.1) were subcutaneously implanted into the mice for 1 week, 4 weeks and 3 months. Before surgery, 1.5% isoflurane in oxygen were used to anesthetize the mice. After shaving, a longitudinal incision was made on the either side of the dorsal region and two hydrogel samples were implanted respectively, wound clips were used to close the incisions. After implantation, all the mice were observed daily and housed for 1 week, 4 weeks and 3 months. In each group, 6 samples of each hydrogel formulation were implanted into three different mice for further statistical significance studies. Histological analysis. At the predetermined time point, mice were sacrificed through CO2 asphyxiation. Subsequently, the implanted samples and their surrounding tissue were collected with scissors, and then fixed overnight using 4% paraformaldehyde at 4 °C. After dehydrated in 30% sucrose solutions, the implants were embedded in optimal cutting temperature (OCT) compound at -20 °C and then cut into 6-µm sections. For histological analysis, the hematoxylin


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& eosin (H&E) staining was used to evaluate the inflammatory response to the implants after 1 week implantation, dark purple and pink indicated the nuclei and cell cytoplasm respectively. Masson’s trichrome staining was used to examine the collagen formation and cell infiltration, in this method, the collagen was stained in blue, the nuclei was stained in black and the cytoplasm was stained in red. All images were observed and acquired using an inverted optical microscope. Macrophage mitigation and infiltration were studied using immunofluorescence stain. The sections were rinsed with PBS for 2 times, and then incubated in 5% normal donkey serum (Jackson Immuno Research Laboratories, Inc., USA) for 30 min at room temperature. The panmacrophage expression in the samples was estimated by incubating the sections with the rat antiF4/80 (dilution 1:50, Abcam, USA) primary antibody overnight at 4°C and then stained by fluorescent secondary antibody (Alexa Fluor 594 donkey anti-rat IgG, Invitrogen, USA) in 1:200 dilution for 2 h at room temperature. The sections were further stained and mounted by the 4,6diamidino-2-phenylindole (DAPI) Fluoromount-G (Southern Biotech, UK) before imaged through fluorescent microscope. Sections stained without the primary antibody were used as the negative control. Results and discussion Preparation of Alg/PEI polyelectrolyte hydrogels As shown in Figure. 1a, the negatively and positively charged polyelectrolytes could selforganize and self-assemble into bulk hydrogel materials driven by the electrostatic interactions. It was also shown that the overall charge of the hydrogels could be tuned by changing the ratio of oppositely charged polyelectrolytes.


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Polyelectrolytes are a large group of charged polymers with either positively or negatively charged groups. Alginates are one type of most widely used negatively charged polyelectrolytes consisted of β-D-mannuronic acid (M block) and α-L-guluronic acid (G block) units (Figure. 1b). 17,18

PEIs are one type of most widely used positively charged polyelectrolyte polymers with lots

of protonated ammonium groups (Figure. 1c).19 In this work, five different formulations of Alg/PEI polyelectrolyte hydrogels (Alg/PEI0, Alg/PEI0.025, Alg/PEI0.045, Alg/PEI0.06, and Alg/PEI0.1) were successfully prepared by mixing alginate and PEI with different ratios and further crosslinked by divalent cation (Ca2+) at room temperature. All the hydrogels were not cytotoxic and passed the endotoxin tests (Supplementary Fig.S1). The zeta potential results of these five hydrogels were -9.5±1.9, -5.3±0.1, -0.5±0.9, 20.8±1.0 and 32.3±2.9 mV, respectively (Figure. 2a). Notably, the zeta potential of Alg/PEI0.045 hydrogel was close to zero, indicating the balanced charged hydrogel could be successfully achieved when the alginate: PEI was 1:0.045.


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Figure 1. a) The formation of the hydrogels based on oppositely charged polyelectrolytes assembly. b) Chemical structure of alginate (the negatively charged polyelectrolyte) and c) branched PEI (the positively charged polyelectrolyte). Characterizations of Alg/PEI hydrogels The water content of the hydrogels was calculated by equation (1). No obvious difference could be observed among the five formulations of hydrogels (Figure. S2a, Supporting information). The Alg/PEI0 hydrogel (pure alginate hydrogel) contained about 95.6% (w/w) of water. While as the increase of PEI, the water content increased slightly, suggesting that the incorporation PEI did not influence the water content of the hydrogels. The compressive modulus of the five formulations was also measured （Figure. S2b, Supporting information）. We found that with the increase of PEI, the mechanical strength of hydrogels decreased from 0.075 MPa to 0.045 MPa (a 40% decrease). It has been demonstrated that the G-blocks content is a critical factor in controlling mechanical strength of alginate hydrogel.17 The incorporation of positively charged PEI could random interact with a considerable amount of carboxylic acid groups (COO-) from either G-blocks or M-blocks. Thus, higher PEI content led to decreased mechanical properties of Alg/PEI hydrogel. Whereas the integrity and uniform structures of the Alg/PEI hydrogels were not affected. Protein adsorption tests Protein adsorption is considered to be the first step when the implants enter the body and determines their fate in vivo. Therefore, to investigate the antifouling properties of the hydrogels, protein adsorption tests were performed at first. As shown in Figure. 2a and 2b, we found that no obvious green fluorescence (FITC-BSA) could be observed on the balanced charged Alg/PEI0.045


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hydrogel, indicating its ability to efficiently resist the BSA adsorption. In contrast, both positively and negatively charged Alg/PEI hydrogels showed strong fluorescence adsorption. The higher of the net charge, the stronger of the fluorescent BSA could be observed. Interestingly, the negatively charged BSA could also adsorb on the surfaces of negatively charged hydrogels. This phenomenon could be caused by the electrostatic interactions formed between the positive charged residues in BSA and the negatively charged alginate in the hydrogels.20 Electrostatic and hydrophobic interactions are the major driven forces of protein adsorption.21 Like antifouling zwitterionic hydrogels, the balanced charged hydrogel is a neutral and hydrophilic material, so it can efficiently eliminate both the electrostatic and hydrophobic interactions with the proteins (Figure. 2c). Thus, this balanced charged hydrogel could significantly resist BSA adsorption and further confirmed our hypothesis that the antifouling hydrogels can be achieved by balancing the oppositely charged polyelectrolyte polymers.


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Figure 2. Protein adsorption on the hydrogels. a) Fluorescence microscopy images of the BSA adsorption. b) Normalized integrated fluorescent intensity. Green: FITC labeled BSA; scale bar = 50 µm. c) Illustration of the different interactions between the proteins and the hydrogels. Cell adhesion tests The antifouling properties of the hydrogels were further evaluated with cell adhesion tests. NIH-3T3 cells were used in this work and the results were shown in Figure. 3a and 3c. Notably, there was no cell adhesion on the surface of balanced charged Alg/PEI0.045 hydrogel. By contrast, as the overall charge deviated from neutral, the cells adhesion significantly increased and the most positively charged Alg/PEI0.1 hydrogel showed the maximum cells adhesion (~68.7%). It could be found that the trend of cell adhesion results was very similar with the protein adsorption tests. This is because the antifouling hydrogel has no interactions with the proteins in the medium or the membrane proteins on the cells. In comparison, the unbalanced charged hydrogels tended to adhere the proteins or the cells which could further bind to the cell membrane proteins (e.g. integrins).22 Since alginate inherently lacks mammalian cell-adhesivity, very few rounded NIH-3T3 cells adhered on the alginate hydrogel surface and some aggregated together in the pore of the hydrogel.


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Figure 3. Cell adhesion tests for the hydrogels. a) Bright field images of NIH-3T3 cells adhered on the hydrogel samples. b) Fluorescence images of live/dead stained NIH-3T3 cells. c) Quantitative data of cell adhered on the hydrogels. d) The viability of the cells adhered on the surfaces of the hydrogels. N.D.: not detected, scale bar = 50 µm, n = 5. Hemocompatibility Hemocompatibility is also a highly desirable property for biomedical materials. However, the adsorption of fibrinogen on blood-contacting biomaterials will result in platelets activation and aggregation, and may further induce thrombotic response, which may be lethal to the patients receiving blood dialysis or blood perfusion.23,24 In this study, whole blood was used and hemolysis assays were performed (Figure. 4). Firstly, the blood cells adhesion on the hydrogels


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was observed after incubation in the fresh blood for 30 min. As expected, it could be clearly found that no blood cell or blood clotting adhered on the balanced charged Alg/PEI0.045 hydrogel, indicating this antifouling hydrogel did not adsorb fibrinogen and trigger the blood clotting. In contrast, the unbalanced charged hydrogel exhibited different degrees of blood cells adhesion and clotting (Figure. 4a). Next, the hemolysis rate (HR) of the hydrogels was quantitatively evaluated. For clinical applications, the HR value must be lower than 5%.16,25 As shown in Figure 4b and 4c, the supernatants of the balanced charged Alg/PEI0.045 hydrogel showed the lowest hemolysis effect and the HR was 0.03%. In comparison, the positively charged Alg/PEI0.1 hydrogel showed the highest hemolysis effect and the HR was 1.15%. Because the excessively positive charge of the hydrogel could damage the RBC membranes and cause the leak of hemoglobin. The above results suggested that all Alg/PEI hydrogels showed low degree of hemolysis, meanwhile the balanced charged Alg/PEI0.045 hydrogel exhibited the best hemocompatibility.

Figure 4. Hemocompatibility assay for the hydrogels. a) Photographs of blood cells adhesion on the surfaces of Alg/PEI hydrogels. b-c) Hemolysis assay for the hydrogel samples.


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Bacterial adhesion tests Then, we studied the anti-bacterial adhesion properties of the hydrogels. Nosocomial infection is a major challenge in clinical practices. This is because bacteria adhesion and associated biofilm formation on medical devices surfaces can lead to infections and more severe complications. Severe E. coli adhesion could be observed on both negatively and positively charged hydrogels, while negligible E. coli colonies could be found on the balanced charged hydrogel (Figure. S3, Supporting information). In many cases, bacteria adhesion also depends on surface adsorption of polysaccharides, which can lead to electrostatic interaction and hydrogen bonging between bacteria the hydrogels.

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Therefore, this result proved that the balanced

charged hydrogel could also efficiently resist polysaccharide adsorption, and thus eliminate bacteria adhesion or biofilm formation, which is highly favorable in the preparation of implantable medical devices or materials. In vivo implantation Along with the remarkably excellent antifouling properties of the hydrogels, the inflammatory responses and capsule formation of the hydrogels were investigated in a mouse model. All the hydrogels passed the endotoxin test and achieved the criteria of in vivo implantation (data not shown). Negatively charged (Alg/PEI0), balanced charged (Alg/PEI0.045) and positively charged (Alg/PEI0.1) hydrogels were subcutaneously implanted in BALB/c mice and retrieved at the predetermined time points (1 week, 4 weeks and 3 months). H&E and Masson’s trichrome stains were used to evaluate the cell infiltration and collagen deposition around the hydrogel samples. As shown in Figure. 5a and 5b, the inflammatory responses to the implants were evaluated 1 week after implantation. The representative H&E and Masson’s trichrome-stained images


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revealed that both the unbalanced charged hydrogels could lead to the accumulation of inflammatory cells and a thick inflammation layer could be observed surrounding the positively charged hydrogel. This was due to the excessively positive charges of the hydrogels could provoke severely acute inflammatory responses. Conversely, negligible amount of inflammatory cells could be observed at the interface between tissue and balanced charged hydrogel. These results suggested that implantation of the balanced charged hydrogel induced much less inflammatory response than those two unbalanced charged hydrogels, which may be due to its superior protein resistant and anti-cell adhesion properties.

Figure 5. The inflammatory responses and capsule formation of the hydrogels after subcutaneously implanted in mice. a) H&E stain images of hydrogel samples after 1 week


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implantation. b-d) Masson’s trichome stain images of hydrogel samples at each time point after implantation. Collagen capsules were stained in blue, red arrow indicates the fibrous capsule, scale bar = 100 µm. Acute inflammatory to implants normally subsides by 3 weeks, and then the collagen capsule begins to form. In this work, the collagen capsule formation surrounding the implants was studied at 4-week post-implantation, and long-term performance of the implants was evaluated at the 3-month post-implantation. As shown in Figure. 5c and 5d, a thick fibrous capsule could be found surrounding the positively charged hydrogel 4 weeks after implantation, and at 3 months, it was still encapsulated by a denser capsule (97.1±9.6 µm). The negatively charged hydrogel pure alginate was also bordered by a capsule （49.9±8.1 µm）at 4 weeks and 3 months, which was consistent with previous literature reports.28 Remarkably, no fibrous capsule was found surrounding the balanced charged hydrogels at both 4 weeks and 3 months. The hydrogel was surrounded by the loosely distributed collagen which was similar with normal subcutaneous tissues, and no cell infiltrated into the hydrogel. These findings proved that the balanced charged hydrogel was stable and could efficiently resist the FBR for at least 3 months, thanks to its excellent antifouling properties. Immunohistochemical analysis Macrophages have been recognized as the key commander of the FBR and orchestrate the immune responses by releasing cytokines and chemical mediators to recruit other cells (e.g. fibroblasts)29. Therefore, to further examine the FBR against three hydrogels, the macrophage behaviors on the tissue-hydrogel interface were investigated 1 week, 4 weeks and 3 months after implantation. In this study, macrophage cells were stained in red and cell nuclei were labeled


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blue. Representative fluorescence images were shown in Figure. 6. At 1 week, a big mass of macrophages could be found at the tissue-hydrogel interfaces and also inside the unbalanced charged hydrogels. In comparison, very few macrophages could be observed at the tissuehydrogel interface of balanced charged hydrogel. Notably, as expected, negligible macrophage could be observed at the interface of tissue-hydrogel of balanced charged hydrogel 4 weeks and 3 months after implantation, whereas macrophages could still be detected at the tissue-hydrogel interfaces of both unbalanced charged hydrogels. These results were consistent with the histological analysis and confirmed at the cellular level that the antifouling balanced charged hydrogel could efficiently evade macrophage recognition and attack, which was the key to avoid the FBR cascade.


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Figure 6. Macrophage migration and infiltration. Representative fluorescence images of panmacrophages biomarker (F4/80) labelled macrophage cells 1 week, 4 weeks and 3months after implantation. Macrophages were stained in red. Scale bar = 100 µm, dotted line indicated the interfaces of tissue and hydrogel, asterisk indicated the hydrogel samples. Conclusions In this study, basing on the balanced charged strategy, we successfully developed a novel hydrogel material which could be easily prepared by balancing the oppositely charged polyelectrolytes. Comparing with its unbalanced charged counterparts, this balanced charged hydrogel not only possessed excellent antifouling properties in vitro, but also could effectively mitigate the FBR and efficiently inhibit the capsule formation for at least 3-month subcutaneous implantation in a mouse model. Other merits of this novel strategy include abundance of raw materials, simplicity of fabrication, availability of functional groups, etc. These merits enable this novel antifouling hydrogel to be an attractive material for various biomedical applications where FBR should be avoided. Acknowledgements The authors acknowledge the ﬁnancial support from the National Natural Science Funds for Excellent Young Scholars 21422605, National Natural Science Funds for Innovation Research Groups 21621004, Tianjin Natural Science Foundation, 14JCYB-JC41600, the Qingdao National Laboratory for Marine Science and Technology, QNLM2016ORP0407. Supporting Information: cytotoxic of the hydrogels, water content, mechanical property and anti-bacterial adhesion property of the Alg/PEI hydrogels.


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Author Information Corresponding Author *E-mail: [email protected] ORCID Lei Zhang: 0000-0003-3638-6219 Notes: The authors declare no competing financial interest.

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PEI Polyelectrolytes Hydrogel

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