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[Debabrata Palai](https://orcid.org/0000-0003-1192-6143), Miho Ohta, Iga Cetnar, [Tetsushi Taguchi](https://orcid.org/0000-0003-2541-2530), [Akihiro Nishiguchi](https://orcid.org/0000-0002-3160-6385)

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[Enhanced ROS scavenging and tissue adhesive abilities in injectable hydrogels by protein modification with oligoethyleneimine](https://mdr.nims.go.jp/datasets/ed970860-640a-4cad-a22c-c745fcfa1910)

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Enhanced ROS scavenging and tissue adhesive abilities in injectable hydrogels by protein modification with oligoethyleneimineBiomaterialsSciencePAPERCite this: Biomater. Sci., 2024, 12,2312Received 20th December 2023,Accepted 11th March 2024DOI: 10.1039/d3bm02065grsc.li/biomaterials-scienceEnhanced ROS scavenging and tissue adhesiveabilities in injectable hydrogels by proteinmodification with oligoethyleneimine†Debabrata Palai, a Miho Ohta,a Iga Cetnar,a,b Tetsushi Taguchi *a andAkihiro Nishiguchi *aPostsurgical treatment comprehensively benefits from the application of tissue-adhesive injectable hydro-gels, which reduce postoperative complications by promoting wound closure and tissue regeneration.Although various hydrogels have been employed as clinical tissue adhesives, many exhibit deficiencies inadhesive strength under wet conditions or in immunomodulatory functions. Herein, we report the devel-opment of reactive oxygen species (ROS) scavenging and tissue-adhesive injectable hydrogels composedof polyamine-modified gelatin crosslinked with the 4-arm poly (ethylene glycol) crosslinker. Polyamine-modified gelatin was particularly potent in suppressing the secretion of proinflammatory cytokines fromstimulated primary macrophages. This effect is attributed to its ability to scavenge ROS and inhibit thenuclear translocation of nuclear factor kappa-B. Polyamine-modified gelatin-based hydrogels exhibitedROS scavenging abilities and enhanced tissue adhesive strength on collagen casing. Notably, the hydrogeldemonstrated exceptional tissue adhesive properties in a wet environment, as evidenced by its perform-ance using porcine small intestine tissue. This approach holds significant promise for designing immuno-modulatory hydrogels with superior tissue adhesion strength compared to conventional medicalmaterials, thereby contributing to advancements in minimally invasive surgical techniques.IntroductionInjectable hydrogels have been used as medical tissueadhesives for effective wound closure, bleeding control, andtissue sealing, thereby contributing to the prevention of post-operative complications.1–3 Although conventional treatmentsusing sutures and staples have been applied to tissue damagein surgery and traumatic injury, these methods have certainlimitations in blood leakage by suture failure, possibility ofbacterial infection, and difficulty in the treatment of softtissues.4 In addition, sutures and staples do not possess bio-logical functionalities to control the tissue response duringthe wound healing process. Injectable hydrogels could beadvantageous for critical treatments by alleviating the risk ofdamaging delicate tissues and providing greater control overwound healing processes.5,6 As biological tissue is a wet andsoft material with layers of interfacial water, which requireextra effort to anchor adhesive materials7,8 various approaches,including chemical reaction-based anchoring and physicalinteractions (hydrophobic interaction,9–12 van der Waalsforce,13 and hydrogen bonding14,15) offer means to enhanceadhesion strength without compromising biocompatibility.Immunomodulation is a critical concept in the realm ofbiomaterials, which depicts a material’s capacity to interfacewith living entities while mitigating adverse effects, such asinjury, toxicity, or rejection by the host’s immune system.16 Inthe acute phase, the inflammatory response retaliates againstpathogens and fungal infections as per the in-body defensemechanism. During the inflammatory phase, pro-inflamma-tory cytokines are secreted by immune cells, inducing the pro-duction of reactive oxygen species (ROS), such as hydrogenperoxide (H2O2), hydroxyl radicals, and superoxide anion rad-icals. Although ROS is a necessary mediator for tissue regener-ation, excessive ROS generation often overwhelms native anti-oxidant activities and nourishes inflammatory responses17,18leading to delayed wound healing. To date, tissue-adhesiveinjectable hydrogels, such as fibrin glue19,20 andcyanoacrylates,21,22 have been used clinically, and ROS-scaven-ging hydrogels23 have been studied; however, it is still challen-ging to develop injectable hydrogels with both sufficient tissue†Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3bm02065gaResearch Center for Macromolecules and Biomaterials, National Institute forMaterials Science, Namiki 1-1, Tsukuba, Ibaraki 305-0044, Japan.E-mail: nishiguchi.akihiro@nims.go.jp, taguchi.tetsushi@nims.go.jpbFaculty of Materials Science and Engineering, Warsaw University of Technology,Al. Waszyngtona 4/8 Warsaw, Poland2312 | Biomater. Sci., 2024, 12, 2312–2320 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View Issuehttp://rsc.li/biomaterials-sciencehttp://orcid.org/0000-0003-1192-6143http://orcid.org/0000-0003-2541-2530http://orcid.org/0000-0002-3160-6385https://doi.org/10.1039/d3bm02065ghttps://doi.org/10.1039/d3bm02065ghttps://doi.org/10.1039/d3bm02065ghttp://crossmark.crossref.org/dialog/?doi=10.1039/d3bm02065g&domain=pdf&date_stamp=2024-04-24http://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065ghttps://pubs.rsc.org/en/journals/journal/BMhttps://pubs.rsc.org/en/journals/journal/BM?issueid=BM012009adhesiveness and ROS scavenging toward immunomodulatoryfunctions to facilitate wound healing.To functionalize the injectable hydrogels, we focused onpolyamines that possess multiple amino groups. Biogenicpolyamines, including spermine and spermidine, play crucialroles in cellular proliferation, differentiation, and signalling incytosol.24 While the precise functions of polyamines remainonly partially understood, they exhibit anti-inflammatory pro-perties, including suppression of excessive ROS generation,inhibition of pro-inflammatory cytokines, prevention of neu-trophil translocation, and inhibition of macrophageformation.25–28 We have previously reported that branchedoligoethyleneimine (bOEI) possesses anti-inflammatory func-tion via ROS scavenging and bOEI-modified hyaluronic acidrecovered ulcerative colitis in mice.29,30 While synthetic polya-mines are extensively used to treat inflammatory diseases, thedevelopment of a tissue-adhesive hydrogel incorporating anti-inflammatory synthetic polyamines with precisely tunedmechanical strength has not been reported.In this study, we fabricated a bOEI-modified gelatin-basedinjectable hydrogel cross-linked with N-hydroxy succinimide(NHS)-terminated 4-arm poly(ethylene glycol) (4-armPEG-NHS) (Fig. 1). Under physiological pH conditions, bOEI isexpected to provide excess amino groups, facilitating rapidcross-linking with the 4-arm PEG-NHS. We studied the effectsof cross-linking between bOEI and different concentrations ofPEG-NHS on the mechanical and adhesive properties of thegel in comparison with those of the collagen casing (mem-brane). This synthetic hydrogel, with a large number ofprimary amine groups from polyamines, has the potential tosuppress macrophage activation by scavenging excess ROS.The findings of this study may contribute to the design ofROS-scavenging and highly tissue-adhesive injectable hydro-gels, enabling minimally invasive surgery and preventing post-operative complications.Experimental sectionMaterialsPorcine skin-derived G was purchased from Nitta Gelatin(Tokyo, Japan). bOEI (Mw = 300 and 600 Da) was purchasedfrom Junsei Chemical Co., Ltd (Japan). NHS-terminated 4-armPEG (Mw = 20 000 Da) was purchased from NOF Corporation(Japan). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydro-chloride (EDC), WST-8 cell counting kit, and 4′6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased fromDOJINDO (Japan). NHS and phosphate buffered saline (PBS)were purchased from Nacalai Tesque (Kyoto, Japan). Enzymelinked immunosorbent assay (ELISA) for mouse TNF-α was pur-chased from R&D systems (USA). 2-(N-Morpholino) ethanesulfo-nic acid (MES), RPMI 1640 (R8758), fetal bovine serum (FBS),and phalloidin with rhodamine were purchased from Sigma-Aldrich (USA). 2′,7′-Dichlorodihydrofluorescin diacetate(DCF-DA, OxiSelect) was purchased from Cell Biolabs, Inc.(USA). Lipopolysaccharide (LPS, E. coli) and paraformaldehyde(PFA) were purchased from Fujifilm Wako Pure ChemicalCorporation (Japan). Dialysis membranes (molecular weight cut-off value: 12 000–14 000) were purchased from Repligen (USA).The mouse macrophage colony-stimulating factor (M-CSF) waspurchased from Mitenyi Biotec (Munich, Germany). NF-κB p65antibody (F-6) was purchased from Cell Signaling Technology(USA). The mouse fibroblast cell line (L929) was purchased fromRIKEN (Wako, Japan).Synthesis of GbOEI polymerTo synthesis GbOEI-2, 1 wt% of G was dissolved in 100 mL of0.1 M MES buffer (pH = 4.8) for 1 h at 50 °C. bOEI (Mw = 300Da, 3.7 mg) was dispersed in 1 mL of buffer and slowly addedto the solution. EDC (238 mg, 1.24 mmol) and NHS (143 mg,1.24 mmol) were added to the solution and stirred for 24 h at50 °C. Finally, GbOEI-2 was obtained after dialysis and freeze-drying the sample for 3 d. The number of amino groups inGbOEI was estimated using the 2,4,6,-trinitrobenzensulfonicacid assay. To synthesize the different types of GbOEIs,different feeding ratios of bOEI, EDC, and NHS equivalents tothe carboxyl groups were used.Preparation of BMDMsAll mouse experiments were approved by the Animal Care andUse Committee of the National Institute for Materials Science(no. 58-2019-2). The detailed procedure for BMDM cell prepa-ration is described elsewhere.29 In brief, mice (female, C57BL/6J, 7-week-old) were euthanized, and the femur and tibia werecollected and cleaned with 70% ethanol for 1 min. Bones werewashed with RPMI 1640 medium (supplemented with 10%FBS and 1% P/S), and bone marrow cells were collected byflushing the medium using a 25-gauge syringe. After filtrationthrough a 70 μm filter and hemolysis with ACK lysing buffer,cells were collected via centrifugation at 1300 rpm for 5 min,washed, and resuspended in differentiation medium(RPMI1640 medium with 10% FBS, 1% P/S, and 40 ng mL−1 ofM-CSF). Cells were cultured for 5 d at 37 °C in a 5% CO2 incu-bator, and differentiated cells were harvested using a scraperfor further experiments.Cell viability assayBMDM (3 × 104 cells per well) cells, and L929 cells (1 × 104)were seeded in a 96-well cell culture plate and cultured indifferentiation medium at 37 °C in an incubator with 5% CO2for 24 h. The differentiation medium containing GbOEIs(100 μg mL−1) and LPS (100 ng mL−1) were then added to thecells and incubated for 24 h. Cell morphology was monitoredusing an optical microscope (EVOS XL Cell Imaging System,Thermo Fisher Scientific, USA) during the incubation. A cellcounting kit (WST-8 assay; DOJINDO, Japan) was used tocount the number of cells. Briefly, 10 µL of WST-8 reagent wasadded to each well and incubated for 2 h. Absorbance at450 nm was recorded using a microplate reader. Cell numberswere calculated using a standard curve.Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 2312–2320 | 2313Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065gELISAInflammatory cytokines were quantified using an ELISA kitaccording to the manufacturer’s protocol. BMDM (3 × 104 cellsper well) cells were seeded in a 96-well cell culture plate andcultured in the differentiation medium for 24 h.Differentiation medium containing GbOEI samples (100 μgmL−1) and LPS (100 ng mL−1) was added to the cells and incu-bated for another 24 h. To measure the inflammatoryresponses of BMDMs, the supernatants were collected carefullyand stored in a −80 °C freezer until use. The secretion oftumor necrosis factor-α (TNF-α) from BMDMs was quantifiedusing an ELISA kit. The absorbance at 450 nm was recordedusing a microplate reader, and the concentrations were calcu-lated from the standard curve.Immunofluorescence stainingFor immunofluorescence staining, BMDMs were cultured onglass-bottom chamber slides (Matsunami, Japan) in a differen-tiation medium and incubated at 37 °C in an incubator with5% CO2. The differentiation medium with or without LPS(100 µg mL−1) and media containing G, GbOEI samples withLPS (100 µg mL−1) were added to the cells and incubated foranother 24 h. The cells were fixed using 4% PFA and permeabi-lized with 0.2% Triton-X for 15 min. After washing with PBSthrice, the cells were blocked with 5% BSA/PBS for 1 h. Thecells were then stained using phalloidin with rhodamine andincubated for 1 h at 25 °C, to visualize the actin filaments. Thecells were further incubated with anti-mouse NF-κB antibody(1 : 100) for 16 h at 4 °C, followed by incubation with a second-Fig. 1 Schematic presentation of the preparation of branched oligoethyleneimine-modified gelatin (GbOEI)-based tissue adhesive and their appli-cation. (a) Synthesis of GbOEI. (b) Schematic of crosslinking between GbOEI and 4-arm PEG-NHS, where ROS scavenging and tissue adhesive abil-ities of injectable hydrogel were improved by bOEI modification. (c) Demonstration of injectability of the hydrogel using a double barrel spiralneedle. Acid blue was used for the visualization of hydrogels.Paper Biomaterials Science2314 | Biomater. Sci., 2024, 12, 2312–2320 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065gary antibody (1 : 500) for 1 h. After washing with PBS, thenuclei were stained with DAPI. Samples were observed using aconfocal laser scanning microscope (CLSM; ZEISS LSM 900,Germany).ROS scavenging assayROS scavenging test of gelatin and the hydrogel was performedusing the fluorescent probe DCF-DA, which is highly sensitiveto hydroxyl radicals. DCF-DA was dissolved in ethanol anddiluted with PBS. To assess ROS scavenging of polymer,DCF-DA solution was added to PBS containing G and GbOEI(100 µg mL−1), and H2O2 was added then in a light protective96-well plate. For the hydrogels, pre-gel solution (50 µL) wasadded to 96-well plate and incubated for 1 h to complete thegelation. H2O2 (50 μL) was then added to the gels followed byincubation for 1 h, and DCF-DA (50 μL) was added. The finalconcentrations of DCF-DA and H2O2 were 50 µM and 100 µM,respectively. Consequently, both the gelatin solution andhydrogels were incubated at 37 °C for 1 h, and fluorescenceintensity with (excitation wavelength 485 nm and emissionwavelength 525 nm) was measured using a microplate reader(Spark10M, TECAN, Switzerland).Rheological measurementRheological measurements were performed using a rheometer(MCR301, Anton Paar GmbH, Austria). G and GbOEI-1, -2, -3,-4 were dissolved in PBS at 10 wt% at 50 °C and kept at 37 °Cin a dry bath until the use. 4-arm PEG-NHS crosslinker wasdissolved in PBS (10, 20, and 30 wt%) at room temperature.Gelatin and PEG-NHS crosslinkers were mixed and placed onthe stage of the rheometer (pre-warmed at 37 °C) using apipettor, and a jig with a diameter of 10 mm was set at a gapof 1 mm. After removing the excess sample, the time-depen-dent shear modulus was measured at an angular frequency of10 rad s−1 with 1% shear strain in oscillatory mode. Hydrogelformation through crosslinking was conformed from the pointwhen both the curve of storage modulus (G′) and loss modulus(G″) reached plateau.Tensile testTensile strengths were measured using a tensile tester instru-ment (EZ-S 500 N, Shimadzu, Kyoto, Japan). A mixture ofGbOEI or G and PEG crosslinker were poured into a dumbbell-shaped silicone mold (total length 35 mm, width 2 mm andthickness 1 mm, respectively). After the incubation at 37 °C for30 min in sealed condition, the gels were removed slowly fromthe mold and fixed with a 1N clamp. The initial distancebetween the clamps was 20 mm. Tensile tests were performedat a speed of 100 mm min−1 as per previous report.31Viscosity measurementG and GbOEI-2 (100 mg mL−1) were dissolved in PBS at 50 °C.Each sample (2 mL) was placed in a plastic tube and incubatedat 37 °C for 20 min. The viscosity of gelatin was measuredusing a rotational viscometer (ViscomateVM-100A, SEKONIC,Japan) by dipping the measuring probe into the sample solu-tions. The probe was thoroughly cleaned with deionized waterbetween the measurements of the two different samples.Swelling behaviour of hydrogelThe swelling ratio of G gel and GbOEI gels was measured byimmersing in PBS at 37 °C. G and GbOEI were dissolved in PBS(10 wt%) at 50 °C. 4-arm PEG-NHS crosslinker was dissolved inPBS (10 wt%, 20 wt%, and 30 wt%) at room temperature. Thesolutions were mixed, and the pre-gel solutions were pouredonto a silicon mold to prepare gel sheets with 1 mm thickness.After incubation at 37 °C for 30 min, each gel sheet was cut into8 mm diameter shaped disks. The gel disks were immersed inPBS (pH = 7.4) and incubated for 24 h at 37 °C. After incu-bation, the gels were collected and weighed (Ws). The gels werefreeze-dried for another 24 h and their dry weights weremeasured (Wd). The swelling ratio was calculated as follows:Swelling ratio ¼ ðW s �WdÞ=Wd ð1Þwhere Ws and Wd represent the weight of swelled and driedgels, respectively.Degradation test under physiological conditionsGbOEI-2 (10 wt%) and 4-arm PEG-NHS (30 wt%) were dissolvedin PBS at 50 °C the and pH of the solution was adjusted. Threemilliliters of the crosslinked solution was added to a siliconemold and a 1 mm thick sheet. Using a punch, 5 mm diameterdisk was prepared and submerged in PBS for 24 h at 5 °C in sealcondition for swelling. The swollen hydrogels were taken intubes and 1 mg mL−1 of collagenase (120 U mL−1, NacalaiTesque, Inc., Japan) was added and incubated at 37 °C. Afterincubation for 1, 4, 8, and 24 h, the supernatants were discarded,and the resulting gels were freeze-dried for 1 d and weighed.Burst strength measurementBurst strength measurements of the gel were conducted accord-ing to the standard protocol of the American Society (ASTMF2392-04).32 As a tissue model, collagen casing (Nippi, Tokyo,Japan) was used as the tissue model. To measure burst strength,the collagen casing was cut to a diameter of 30 mm with a3 mm pinhole. Gelatin and 4-arm PEG-NHS were dissolved aspreviously described. The effect of different concentrations (10,20, and 30 wt%) of 4-arm PEG-NHS on the burst strength of thegel was studied. The pre-gel solutions were poured on the col-lagen casing of 15 mm diameter and 3 mm pinhole, supportedwith silicone disc (1 mm in thickness and 15 mm of an innerdiameter) and incubated at 37 °C for 30 min in sealed con-dition. The collagen casing with the cross-linked hydrogel wasthen placed onto the burst strength measurement apparatus,and saline flowed underneath the sample at 2 mL min−1. Themaximum pressure at which the hydrogel ruptured, either side-wise or in the middle, was recorded.Tissue adhesion of hydrogels to small intestineTo evaluate the adhesive properties of the GbOEI-2 hydrogel,ex vivo models of the porcine-derived small intestine (TokyoBiomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 2312–2320 | 2315Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065gShibaura Zouki, Japan) were used. First, the intestines werecleaned and filled with saline solution. A 3 mm puncture wascreated at the center of the intestine using a biopsy punch(KAI Medical, Japan), and the intestine was held vertically toensure leakage of saline from the puncture. The excess waterwas wiped off and the hydrogel was applied, and the adhesiveproperties of the hydrogel were ensured by applying pressure.Results and discussionSynthesis of GbOEI and their anti-inflammatory functionsTo address whether the modification of gelatin with bOEI pro-vides anti-inflammatory properties, we prepared four differentGbOEIs with different numbers of amino groups via EDC/NHSchemistry (Table 1). To prepare different types of GbOEIs, theratios of EDC to NHS were varied. The contents of aminogroups were in the range 501–926 μmol g−1 after the modifi-cation, and GbOEI-2 showed a 3.3-fold increase in aminogroups compared to non-modified G. The introduction ofbOEI into GbOEI-2 was characterized using 1H-NMR (Fig. 2a).Next, cytotoxicity of GbOEI was evaluated using cell culturemodels of mouse fibroblast cell line (L929 cells) and mouseprimary macrophages (BMDMs). L929 cells were exposed tosupernatants obtained from each hydrogel after 24 h of incu-bation. Live/dead, WST-8, and wound healing assay showedthat hydrogels were biocompatible and did not inhibit cellularmigration (Fig. S1†). After 24 h of incubation of BMDM cellswith the four types of gelatin (GbOEI-1,-2,-3, and -4) solutions,GbOEI-2 showed the highest cell viability and was not signifi-cantly different from G (Fig. 2b). In contrast, the GbOEI-3 and-4 samples showed lower cell viability than the others, partiallydue to intra/intermolecular crosslinking with bOEI and thetoxicity of bOEI600. As an Inflammation model, BMDMs getstimulated by LPS which binds to LPS binding protein to forma complex with the myeloid differentiation factors 2 and CD14connecting to Toll-like receptors (TLRs), leading to the acti-vation of NF-κB pathway and initiating production of pro-inflammatory cytokines such as TNF-α.33–35 To evaluate anti-inflammatory function of GbOEI, the secretion of TNF-α fromLPS-stimulated BMDMs was quantified. ELISA result impliesthat GbOEI-2 substantially supressed TNF-α secretion to ahigher extent in LPS-stimulated BMDMs, without jeopardizingcell viability (Fig. 2c). The secretion of TNF-α per individualcell was the most effectively suppressed by GbOEI-2, highlight-ing the capacity of GbOEI-2 to mitigate cytokine secretion atthe level of individual cells (Fig. 2d). GbOEI-4 demonstrated acomparable ability to suppress cytokine release; however, thedecrease in cell viability made it a less favorable choice. Tounderstand the anti-inflammatory mechanisms, we furtherevaluated the capability of GbOEI-2 to impede the nucleartranslocation of NF-κB in LPS-stimulated BMDMs. AlthoughbOEI is a positively charged molecule, a previous report hasindicated that it does not induce protein aggregation or LPSinactivation through oppositely charged interactions.Moreover, it exhibited highest anti-inflammatory functions byblocking a broad spectrum of innate immune response andinhibiting the NF-κB/activator protein-1 pathways.29 CLSMFig. 2 Anti-inflammatory property of GbOEIs. (a) 1H NMR of G andGbOEI-2 (DMSO, 400 MHz). (b) Cell viability of BMDMs exposed tomedia, LPS, G and GbOEI solutions (n = 3). (c) The amount of TNF-αsecreted from LPS-stimulated BMDMs in the presence of G and GbOEI(n = 3). (d) The amount of TNF-α secretion per 1 × 104 cells (n = 3).(e) CLSM image of BMDMs exposed to media, LPS, LPS + G, and LPS +GbOEI-2. NF-κB p65 (green) and nuclei (blue) were stained with NF-κBp65 antibody and DAPI, respectively. Data are presented as mean ± s.d.*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, analyzed using theone-way ANOVA, followed by Tukey’s multiple comparison post hoctest. n.s. denotes not significant.Table 1 Synthesis of gelatin with different amount of amino groupsGelatinMolecularweight ofbOEI (Da)Content of aminogroups in gelatin(µmol g−1)Ratio ofequivalents tocarboxyl groupsYield(%)bOEI EDC NHSG — 282 — — — 97GbOEI-1 300 501 10 10 1 108GbOEI-2 300 926 10 1 1 99GbOEI-3 300 627 1 1 1 72GbOEI-4 600 721 5 1 1 97The amino groups in gelatin was calculated by determining theresidual amino groups using 2,4,6,-trinitrobenzensulfonic acid (TNBS).Paper Biomaterials Science2316 | Biomater. Sci., 2024, 12, 2312–2320 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065gobservation also corroborate that GbOEI-2 effectively suppressedthe nuclear translocation of NF-κB in LPS-stimulated BMDMs,while localization of NF-κB in nuclei was partially observed in G(Fig. 2e). These results indicate that GbOEI-2 exerts anti-inflam-matory effects by suppressing the nuclear translocation of NF-κB, which agrees with our previous finding.29ROS scavenging ability of gelatin and hydrogelConsidering the ROS-scavenging abilities of biogenic polya-mines, we postulated that GbOEI might have ROS scavengingability which contribute in anti-inflammatory effects.25Oxidative stress serve a pivotal role in the physiological defensemechanisms of living organisms. However, the overproductionof ROS impedes tissue regeneration in injured tissues andinduces adverse effects, such as inflammation, necrosis, andfibrotic scarring.18 Consequently, biomaterials capable ofscavenging excess ROS at the wound site are integral to promot-ing regeneration by immunomodulation. The ability to scavengehydroxyl radicals was quantified using DCF-DA, which is ordina-rily non-fluorescent but increases fluorescence intensity inresponse to hydroxyl radicals. In contrast to G, GbOEI-2 and -4effectively reduced hydroxyl radicals generated by H2O2, func-tioning as ROS scavengers (Fig. 3a). In agreement with the sper-mine-mediated ROS scavenging mechanism, the quantity ofprimary amines present in GbOEI plays a crucial role.25Moreover, in the GbOEI hydrogel, the primary amine was con-sumed during the crosslinking reaction with 4-arm PEG-NHS,but the GbOEI-2 hydrogel still exhibited ROS-scavenging ability(Fig. 3b). Excessive ROS generation triggers the activation ofupstream kinases, such as IkB kinase (IKK), leading to the phos-phorylation and degradation of the inhibitor protein IkB.34 Thisprocess promotes the nuclear translocation of NF-κB and thetranscription of pro-inflammatory genes. Therefore, the ROS-scavenging ability of GbOEI hydrogels may suppress a series ofexcessive inflammatory responses and function as immunomo-dulatory biomaterials. In our previous investigation we eviden-tially proposed a mechanism of bOEI sample where it supressinflammatory responses by scavenging excessive intracellular orextracellular ROS and inhibiting the nuclear translocation ofNF-κB.29 It also supports the ROS scavenging ability and anti-inflammatory functions of GbOEI hydrogels.Mechanical property of GbOEI hydrogels and injectibilityPEG is a biocompatible polymer widely used to formulate tissue-adhesive materials.36,37 As 4-arm PEG-NHS has high reactivitytowards nucleophilic amino groups, the increase in aminogroups in gelatin may trigger rapid gelation by increasing thecrosslinking points. It is expected that the crosslinked hydrogelwill strongly adhere to tissues through a reaction between theremaining NHS groups and the primary amines of proteins inthe tissue.38 For efficient crosslinking, depending on thenumber of amino groups in G and GbOEI, the concentrations of4 arm PEG-NHS can be varied. In the case of the unmodified Ghydrogel, a high elastic modulus and loss modulus wereachieved at 10 wt% concentration of the 4-arm PEG-NHS (Fig. 4aand b). In contrast, the highest elastic and loss moduli for theGbOEI-2 hydrogel were achieved with 30 wt% 4-arm PEG-NHS.Nevertheless, the storage modulus of the GbOEI-2 hydrogelreached a plateau within a few minutes, which was sufficient tomeet the time required for clinical mixing and injection of thegel.39 Mechanical strength of GbOEI-2, G hydrogel was evaluatedby tensile test. At 25 °C, GbOEI-2 hydrogel exhibited 1.5 foldincrease in fracture strain (245%) and 3.6 fold increase in frac-ture strength (90.4 kPa) compared to G hydrogel (Fig. 4c and d).Furthermore, the swelling ratio of the GbOEI-2 hydrogel was 43,which is a 1.9-fold increase compared with that of the G hydro-gel. To understand the injectability at physiological temperature,viscosity of the GbOEI solutions was measured at 37 °C. The visc-osities of all GbOEI solutions were substantially lower than thoseof G (Fig. 4e). This observation supported the injectability ofbOEI at physiological temperatures. GbOEI solution was liquideven at room temperature (∼25 °C) partly due to chemical struc-tural changes of gelatin by bOEI modification and there was noneed to warm up when used. The biodegradability of hydrogelswas evaluated using collagenase (Fig. 4f). During the incubationof the GbOEI-2 hydrogels in the presence of collagenase, theweight gradually decreased, indicating that the hydrogels wereenzymatically degradable.Tissue adhesive property of hydrogelVarious hydrogel was developed using multiple hydrogen andionic bonds for wearable devices, and human–machine inter-faces but tough tissue adhesive hydrogel synthesized usingconjugation reaction between highly reactive primary amineand 4-arm PEG-NHS can be advantageous for minimally inva-sive surgery.40 The tissue adhesive properties of the GbOEIhydrogels were determined according to the ASTM standardprotocol with the instrumental setup (Fig. 5a). To conduct thetest with maximal efficacy, the hydrogel needs to effectivelyFig. 3 ROS scavenging property. (a) ROS scavenging of G, GbOEI-1, -2,-3, -4 (n = 3). DCF fluorescence of control was set to 100%. (b) ROSscavenging of G hydrogel crosslinked with 10 wt% 4-arm PEG-NHSand GbOEI-2 hydrogel crosslinked with 30 wt% 4-arm PEG-NHS. DCFfluorescence of G hydrogel was set to 100%. Data are presented asmean ± s.d. ****P < 0.0001, analyzed using the one-way ANOVA, fol-lowed by Tukey’s multiple comparison post hoc test for (a) and Student’st-test for (b).Biomaterials Science PaperThis journal is © The Royal Society of Chemistry 2024 Biomater. Sci., 2024, 12, 2312–2320 | 2317Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065ginterface with the underlying collagen casing and manifestmechanical strength that surpasses the physiological loadingconditions. Burst-strength measurements were performed toquantify the adhesive capability of the hydrogel under increas-ing liquid pressure. Sustaining the maximum pressure untilrupture signifies the mechanical resilience of the hydrogel.The results indicated that the GbOEI-2 and -4 hydrogels cross-linked with 30 wt% 4-arm PEG-NHS demonstrated the highestburst strength and exhibited an increase of more than two-foldcompared to that of the G hydrogel (Fig. 5b). For GbOEI-1, -3,optimal strength was attained when these gelatins were cross-linked with 10 wt% and 20 wt% 4-arm PEG-NHS, respectively.Modification with bOEI enhanced the burst strength partlybecause of the improved mechanical strength and anchoringwith proteins in the tissues. However, GbOEI hydrogels cross-linked with 30 wt% 4-arm PEG-NHS exhibited better adhesionstrength and anti-inflammatory properties compared withcommercially available and other reported adhesives(Table S1†). The sealing properties of the hydrogel were investi-gated using the porcine small intestine. Immediately after thepuncture, saline water started draining from the puncture site.After applying the GbOEI-2 hydrogel to the puncture site,the intestine was refilled with saline water, but the draining ofthe saline water was stopped immediately (Fig. 5c). Evenwhen pressure was applied to the puncture site, theGbOEI-2 hydrogel firmly sealed the puncture, indicating thatGbOEI hydrogels can be used to treat wounds under wet con-ditions to prevent postoperative complications.ConclusionIn conclusion, we developed ROS scavenging and tough tissueadhesives achieved through the modification of gelatin with cat-ionic bOEI. Upon comparing various Gs containing differentcompositions of bOEI, it was established that GbOEI-2 exhibitedFig. 4 Rheological property and biodegradability of hydrogels. (a and b)Rheological analysis of G and GbOEI-2 crosslinked with different con-centration of 4-arm PEG-NHS. Time-dependent storage modulus (G’)and loss modulus (G’’) were measured at an angular frequency of 10 rad s−1with 1% shear strain in an oscillatory mode at 37 °C. (c) Tensile test of Gand GbOEI-2 hydrogels. (d) The fracture strain and stress exhibited by Gand GbOEI-2 gel (n = 3). The measurement was performed at 25 °C. (e)The viscosity of gelatin solutions at 37 °C. (f ) Biodegradability test ofGbOEI-2 hydrogels using collagenase under physiological conditions.Data are presented as mean ± s.d. **P < 0.01, ***P < 0.001, ****P <0.0001, analyzed using the one-way ANOVA, followed by Tukey’s mul-tiple comparison post hoc test.Fig. 5 Tissue adhesive property of hydrogels. (a) Schematic of burstpressure measurement. (b) Burst pressure of hydrogels of G andGbOEI-1, -2, -3, -4 crosslinked with 4-arm PEG-NHS (n = 3). (c) Imagesof the adhesion test using small intestine of porcine. Acid blue was usedfor the visualization of hydrogels. Data are presented as mean ± s.d. **P< 0.01 and ****P < 0.0001, analyzed using one-way ANOVA, followed byTukey’s multiple comparison post hoc test. n.s. denotes not significant.Paper Biomaterials Science2318 | Biomater. Sci., 2024, 12, 2312–2320 This journal is © The Royal Society of Chemistry 2024Open Access Article. Published on 13 March 2024. Downloaded on 5/1/2024 12:41:33 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Onlinehttp://creativecommons.org/licenses/by-nc/3.0/http://creativecommons.org/licenses/by-nc/3.0/https://doi.org/10.1039/d3bm02065gthe highest cytocompatibility and anti-inflammatory and ROS-scavenging abilities. GbOEI-2 effectively suppressed thesecretion of inflammatory cytokines in LPS-stimulated BMDMsby ROS scavenging and inhibiting the nuclear translocation ofNF-κB. GbOEI-2 hydrogels cross-linked with 4-arm PEG-NHSdemonstrated an exceptional capacity to mitigate ROS overpro-duction. The viscosity of the GbOEI-2 solution at physiologicaltemperatures remained sufficiently low for injectability. TheGbOEI-2 hydrogel exhibited remarkable tissue adhesion andsealing properties when applied to collagen casings andporcine-derived intestinal tissue. Our findings may promotescarless wound healing and enhance the quality of minimallyinvasive surgeries. This innovation has the potential to reducethe postoperative complications and burden.Conflicts of interestThe authors declare no conflict of interest.AcknowledgementsWe appreciate financial support from the Japan Society for thePromotion of Science (JSPS) KAKENHI (Grant No. 22H03962and 23H01718) and the Uehara Memorial Foundation.References1 B. Petersen, A. Barkun, S. Carpenter, P. Chotiprasidhi,R. Chuttani, W. Silverman, N. Hussain, J. Liu,G. Taitelbaum, G. G. Ginsberg and Technology AssessmentCommittee, American Society for GastrointestinalEndoscopy, Gastrointest. Endosc., 2004, 60, 327–333.2 N. Lang, M. J. Pereira, Y. 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