Bioinspired, Injectable, Quaternized Hydroxyethyl Cellulose

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Biological and Medical Applications of Materials and Interfaces

Bioinspired, Injectable, Quaternized Hydroxyethyl Cellulose Composite Hydrogel Coordinated by Mesocellular Silica Foam for Rapid, Noncompressible Hemostasis and Wound Healing Chengwei Wang, Haoyi Niu, Xiaoyu Ma, Hua Hong, Yuan Yuan, and Changsheng Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b08799 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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ACS Applied Materials & Interfaces

Bioinspired, Injectable, Quaternized Hydroxyethyl Cellulose Composite Hydrogel Coordinated by Mesocellular Silica Foam for Rapid, Noncompressible Hemostasis and Wound Healing Chengwei Wang,a, b, c Haoyi Niu,a, b Xiaoyu Ma,a,b Hua Hong,a, c Yuan Yuan,*a, b Changsheng Liu *a, b a. Key Laboratory for Ultrafine Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China. E-mail: [email protected], [email protected] b. Engineering Research Center for Biomedical Materials of Ministry of Education, East China University of Science and Technology, Shanghai 200237, PR China c. Shanghai Wego Biological Technology Co., Ltd, Shanghai 200237, PR China

Abstract Massive bleeding control and anti-infection are the major challenge for urgent trauma with deep and noncompressible hemorrhage in both clinic and battlefield. Inspired by the coordinated primarily blood clot formation and secondly coagulation cascade activation in natural hemostasis process, an injectable, quaternized hydroxyethyl cellulose (HEC)/mesocellular silica foam (MCF) hydrogel sponge (QHM) for both hemorrhage control and antibacterial were prepared via one-pot radical graft copolymerization. The as-prepared QHMs exhibited instant water-triggered expansion and superabsorbent capacity, and thereby effectively facilitated blood components concentration. Moreover, the QHM1 with appropriate amount of MCF (9.82 w/w %) could further active the coagulation factors. Synergistically, the QHM1 could reduce the plasma clotting time to 59  4 % in vitro, and showed less blood loss than commercial available hemostatics in vivo noncompressible hemorrhage models of lethal rabbit-liver defect. Furthermore, the QHM with quaternary ammonium (QA) groups density of 2.732 mmol/g exhibited remarkable antibacterial activities and excellent cytocompatibility. With the efficient hemostasis efficacy and excellent antibacterial behavior, QHM dramatically facilitated the wound healing in a full1

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thickness skin defect model in vivo. Thus, this QHM represents a promising hemostatic in more widespread clinical application. Keyword: hydroxyethyl cellulose, mesocellular foam, hemostasis, antibacterial, wound healing

1. Introduction Uncontrollable hemorrhage poses significantly fatal risks in battlefield, emergency, and hospital settings and has caused over 5.8 million deaths per year worldwide nowadays.1-2 Concomitantly, wound infection, which often causes delay healing or even nonhealing wounds, is another challenge facing caregivers, especially for the combat trauma wound.3-4 Ideally, an effective hemostatic materials should be endowed with excellent anti-infection function. To date, topical hemostatic agents available in clinic mainly include glutaraldehyde cross-linked BioGlue5, zeolite-based QuikClot6 and chitosan-based HemCon5. Unfortunately, the hemostatic efficacy and anti-infection capacity of these traditional materials often cannot meet the clinic requirement, especially for the deep, irregularly shaped or noncompressible wounds incurred in both clinic and battlefield. According to a spontaneous hemostasis process of the organism, an ideal hemostatic material for massive bleeding should rapidly absorb water, concentrate the blood components, provide a physical barrier to blood flow, and initiate an endogenous or extrinsic coagulation system. Finally, a stable clot is formed to cease bleeding.1, 7-8 From this standpoint, serials of water-absorbing polymeric materials, including chitosan, polyethylene glycol, and their derivatives, have been developed for facilitating the local blood cells aggregation and plasma proteins concentration to accelerate coagulation.2, 9-10 Notably, some novel superabsorbent and shape memory sponge/foams exhibited good hemostatic capability.7,

11

Specifically, the FDA-

approved cellulose sponges-based XStat™ device, which can be injected and rapidly swell to padding cavity and apply pressure, has achieved great success for the control of severe, life-threatening bleeding from narrow entrance extremity wounds.5,

11

However, XStat™ itself is nondegradable and need to take out each sponge from the wound site.1 Also, the water-absorption capacity of the shape-memory polymer foams (SMPF) are often low, and thus it needs decades of seconds to recover to their initial state.11-12 Furthermore, only by rapid water-triggered concentration, the XStat™ and these SMPF are lack of inherently confined coagulation factors and thus show limited hemostasis efficacy.13 Besides above primary formation of blood clot, endogenous or extrinsic coagulation factors activation is another key element for efficient 2


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hemostasis.14-15 It is noteworthy that the mesoporous silica (MS), with porous structure, high specific surface area and high density of polar silanols as well as negative charges, could not only facilitate the local concentration of the blood cells and hemostatically active protein,16 but also activate hageman factor (Factor XII, FXII) of the endogenous coagulation cascade, thus synergistically achieved a rapid blood-controlling.13 This study provides a strategy to realize hemagglutination/ platelet aggregation- and coagulation factors activation-induced coagulation simultaneously. Due to inherent interconnected macroporous structure, and remarkable superabsorbent and shape memory property, hydrogels sponge should be a potential hemostatic agent, especially for deep and irregular hemorrhage. Meanwhile, it is wellaccepted that, to overcome the drawbacks of antibacterial agents loading, for a large, exposed wound area, the hemostatic materials with inherent antibacterial activity to inhibit wound infection is an alternative strategy.12 Because of typically positive charge, quaternary ammonium (QA) group exhibit broad-spectrum and efficient antimicrobial properties.17 Moreover, quaternized polymer could promote fibrinogen and blood cells attachment, and thus facilitate hemagglutination,14, formation,1,

6, 14

18

and seal

which should do favor for the hemostasis. Hydroxyethyl cellulose

(HEC), a sort of non-ionic natural cellulose ether, pose ultrahigh water retention ability, biocompatibility and biodegradability.19-20 Moreover, abundant hydroxyl groups endow HEC with superior water absorbing capacity and easy conjugation and modification.19, 21 These properties suggested HEC, if quaternized and fabricated into hydrogel sponge, is a desirable candidate for anti-microbico, hemostatic material, which has never been reported till now. Herein, we endeavored to develop an injectable, quaternized HEC/ MCF composite hydrogel sponge for effective noncompressible hemostasis and antibacterial. As showed in Scheme 1, the quaternized HEC hydrogel sponge can provide excellent water absorption, swell ability, blood components concentration and primary formation of blood clot as well as antibacterial properties. MCF, a kind of silica-based nanomaterials with opened ultra-large spherical mesopores and large pore volume, could guarantee a direct interaction between the coagulation-promoting proteins and the inner surfaces and thus efficiently activated coagulation factors. Synergistically, the 3



quaternized HEC/MCF hydrogel sponge realized a biomimicking hemostasis process. To avoid the degradation of HEC caused by ammonium persulphate (APS), the QHM hydrogel was synthesized by activation of APS and crosslinking with PEGDA via onepot method. The possibility of the composite as superior hemostatic was assessed in items of their morphology, physicochemical properties, in vitro anti-microbico behavior, plasma coagulation time, as well as hemostasis efficiency in extremely serious bleeding in vivo. Also, MTT, live/dead staining and hemolysis examination were employed to assess the safety and cytocompatibility. In addition, the potential wound healing application was also confirmed.

4


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Scheme. 1 Illustration of the synthesis process and properties of the QHM hydrogel sponge. (A) Synthesis of QHM hydrogel by one-pot process. The precursors (MATMA, HEC, PEGDA) were mixed homogeneously, and then were activated by APS and crosslinked by PEGDA to form QHM hydrogel. (B) Schematic diagram of the formation of MCF loaded composite hydrogel. MCF were introduced into the QHM precursor solution, and mixed homogeneously. Similarly, the mixture was actived by APS and crosslinked by PEGDA. (C) Illustration of the hemostasis and antibacterial process of the QHM hydrogel sponge. The QHM hydrogel 5



sponge was input the wound and the superabsorbent properties of QHM was triggered a rapid swelling and facilitated a concentration of blood components, such red blood cells and platelets. Subsequently, an amount of blood proteins was absorbed via electrostatic interaction, and then the absorbed coagulation factor XII was activated by MCF, which led to an acceleration of clot formation. Eventually, a rapid hemostasis was achieved. Meanwhile, the positive QA group on the quaternized hydroxyethyl cellulose could obstruct the potential infection of microbes and exhibited a desirable antibacterial activity.

2. Materials and Methods 2.1 Materials 2-Hydroxyethyl cellulose (HEC, MV ~720000), Pluronic triblock copolymer P123 (P123, Mn ~5800), Poly (ethylene glycol) diacrylate (PEGDA, Mn 700) and 2(methacryloyloxy)-ethyltrimethylammonium chloride solution (MATMA,75 wt %) were purchased from Sigma Aldrich Chemicals. Tetraethylorthosilicate (TEOS), hydrochloric acid (HCl, 37%), and mesitylene (TMB) were obtained from Shanghai Sinopharm Co. Ltd. Ammonium persulphate (APS), ammonium fluoride (NH4F), and N, N, N’, N’-tetramethylethylenediamine (TEMED) were obtained from Aladdin Industrial Co. Ltd. Standard strains of Staphylococcus aureus (S. aureus, ATCC 25923) and Escherichia coli (E. coli, ATCC 25922) were purchased from China General Microbiological Culture Collection Center. 2.2 Synthesis of Mesocellular Silica Foam According to previously reports,22 40 mL of concentrated HCl and 260 mL of deionized water (DI) were mixed by magnetic stirring, then 16 g of P123 was dissolved into the mixture at 40 C. After that, 16 g of TMB was dropped and stirred for 2h under 40 C. Afterwards, 36 mL of TEOS was added with vigorously stirring. After 8 min, the giving mixture was added to a tetrafluoroethylene autoclave and aged for 24 h under 40 C, after which 184 mg of NH4F was mixed into the solution, and aged for another 24 h under 100 C. The as-prepared product was collected by vacuum filtering, and rinsed with ethanol and DI, drained under 80C, and calcined in air for 6 h under 600 C to remove surfactant. 2.3 Preparation of QHM Hydrogel Sponge According previously reports, radical graft copolymerization was employed to prepare quaternized composite hydrogels.23 Briefly, 1.5 mL of MATMA, 0.5 g of HEC, 6


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0.4 mL of PEGDA, 29 mL of DI and a specific amount of MCF (Table 1) were added into flask, mixed by magnetic stirring, adjusted the pH of the mixture to 7~7.1 and bubbling by argon for 10 min. After that, the solution was heated to 60 C and continuously stirred and bubbled for another 10 min. Then, 50 mg of APS and 15 L of TEMED were added with continuous stirring. After 10 min, the blend was keep at 60 C for 24h. After that, the hydrogels were transferred to DI to purification for 72 h, the water was changed every 12 h. Finally, samples were drained by freeze-drying and stored for further use. Table 1 Composition of MATMA, HEC, PEGDA and MCF for various QHMs preparation. MATMA(mL)

HEC (g)

PEGDA (mL)

MCF (g)

QHM0

1.5

0.5

0.4

0

QHM1

1.5

0.5

0.4

0.15

QHM2

1.5

0.5

0.4

0.3

2.4 Characterization MCF was characterized by transmission electron microscopy (TEM, JEOL, Japan) and Micromeritics ASAP2010 sorptometer (Micromeritics, USA) to confirmed its mesoporous structure. The finally MCF contents in QHMs were confirmed by thermogravimetry differential scanning calorimetry (TG-DSC) thermograms analysis (Madison, WI, USA). The time-dependent rheological properties examination was performed for 15 min under a constant frequency of 1 Hz and a strain of 1% under 60 °C to study hydrogels formation kinetics, and recorded the changes in storage modulus (G') and loss modulus (G") over time. Moreover, the frequency-dependent rheological properties of QHMs in its swollen state were also characterized at a strain of 0.1% under 37 °C. The Fourier-transform infrared (FTIR) spectra of samples was obtained using a Perkin Elmer System 2000 spectrometer (Midac, USA). The morphology of hydrogel sponges was characterized using a Scanning electron microscopy (SEM, JEOL, Japan). The NMR spectra of samples were also tested using a 4-mm MAS probe (Bruker Avance 500 MHz). 2.5 Water Absorption 10 mg of dry specimens were put into pre-weighed plastic tubes, followed by adding of 2 mL of DI, PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4.12H2O, 1.5 7



mM KH2PO4, pH 7.4) or rabbit source platelet poor plasma (PPP, prepared as described 2.7.1), and placed on a bench. After 10 min, the excess fluid in tubes were drained before being weighed. All samples were replicated (n = 3). Swelling percentage was calculated utilizing the following formula: Water absorption(%) =

Mw ― Md Md

× 100%

where Mw and Md are represent the weight of sample in swollen and dry state. 2.6 Cytotoxicity Test 2.6.1 MTT Mouse myoblast cells line (ACTT, C2C12) was employed to investigate the cytotoxicity of the specimens by MTT assay. The specimens do not any affect the MTT assay in Dulbecco's Modified Eagle's Medium (DMEM) only. Briefly, C2C12 cells were cultured in DMEM that changed twice a week. Subsequently, each well of 24 well-plates was seeded 30000 C2C12 cells, and co-cultured in a fully humidified atmosphere of 5% CO2 for 24 h under 37 C. After that, the cells were exposed to samples at concentration of 0.5 mg mL-1 for another 24 h, and DMED only was taken as control group. Then, the viabilities of C2C12 cells were evaluated by MTT assay and calculated by means of at least three wells and presented as viability of cells compared with control group (DMEM cultured only). 2.6.2 Live/Dead Staining Assays Live/Dead staining assay was also utilized to estimate cell viability. Briefly, each dish was seeded about 1 × 105 C2C12 cells that cultured with normal DMEM in a 5% CO2 atmosphere under 37 °C. After incubation for 24 h, the C2C12 cells were exposed to samples for another 24 h. After that, each dish was stained by Live/Dead Staining Kit (ThermoFisher Scientific) after the medium of removing and rinsing 5 times by PBS. 2.7 Hemolysis and in Vitro Coagulation Evaluation  2.7.1 Blood Related Component Collection and Preparation Citrated whole blood (CWB) was prepared by drawing of whole blood from healthy male New Zealand White Rabbit with a ratio of 9:1 for 3.8% sodium citrated. Packed red blood cells (RBC) was obtained by centrifugation of CWB under 3000 rpm for 15 min, and rinsed. After that, 5% hematocrit of RBC suspension was prepared 8


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with 100 μL of packed RBC per 2 mL of PBS. Platelet poor plasma (PPP) was obtained as the supernatant by centrifugation of CWB under 3000 rpm for 15 min. Platelet rich plasma (PRP) was obtained as the supernatant by centrifugation of CWB under 1500 rpm for 5 min. 2.7.2 Hemolysis Assay According previous reports,24 sample in 0.8 mL of PBS at set concentrations was mixed with 0.2 mL of the RBC suspension. PBS and water (0.8 mL) only were utilized as negative and positive control. The blend was co-cultured for an hour under 37 °C, Subsequently, the specimens were isolated by centrifugation and the supernatant absorbance at 541 nm was tested. The hemolysis percentage of RBCs was estimated by the following equation: 𝑝𝑒𝑟𝑐𝑒𝑛𝑡 ℎ𝑒𝑚𝑜𝑙𝑦𝑠𝑖𝑠 (%) =

𝑂𝐷𝑆𝑎𝑚𝑝𝑙𝑒 ― 𝑂𝐷𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 𝑂𝐷𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝑂𝐷𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑐𝑜𝑛𝑡𝑟𝑜𝑙

× 100%

2.7.3 Blood Clot Index (BCI) BCI was calculated as previously reports with some modifications.1, 25 Briefly, 100 μL of CWB was slowly dropped on the surface of samples and further incubated in a thermostatic shaker under 37 °C. After 5 minutes, 20 mL of DI were carefully added in the beaker without perturbing the clotted blood, and commercial available gelatin sponge (GS, Guangzhoushi Kuai Kang Yi Liao Qi Xie CO., LTD) selected as control sample. The blood coagulation test was assessed by spectrophotometric measurement of the relative absorbency of blood sample that had been diluted by 20 mL DI at 541 nm. The absorbency of solution of DI (20 mL) and CWB (100 μL) at 541 nm was assumed to be 100%, which was utilized as reference value. The BCI of different materials was evaluated as the following formula: 𝐵𝐶𝐼 (%) =

𝑂𝐷𝑆𝑎𝑚𝑝𝑙𝑒 𝑂𝐷Reference value

× 100%

2.7.4 RBC Attachment RBC attachment was assessed as previously reports with some changes.18 200 μL of RBC suspension were dropped on 10 mg of sample, incubated under 37 °C. After 1 h, the specimens were washed five times with PBS and transferred into 5 mL DI for another 1 h to lysis the attached RBC. 200 μL of RBC suspension transferred into 5 mL 9



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DI were used as reference value. And commercial available GS selected as control sample. Eventually, 100 μL of lysed liquid were measured under UV-Vis 541 nm. The percentage of adhered RBC was quantified by the following equation: 𝑝𝑒𝑟𝑐𝑒𝑛𝑡 𝑜𝑓 𝑎𝑑ℎ𝑒𝑟𝑒𝑑 𝑅𝐵𝐶 (%) =


× 100%

2.7.5 Platelet Adhesion Platelet adhesion assay was evaluated as previously reports with some modifications.1, 16, 26 10 mg of sample was weighted and placed into 24 well plates, and commercial available GS selected as control sample. After that, 50 μL of PRP was pipetted onto the sample and incubated under 37 °C. After 1 h, unattached platelets were gently rinsed five times by PBS. Thereafter, 0.6 mL 1% Triton X-100 in PBS was utilized to lyse the attached platelets for 1h under 37 °C. The lactate dehydrogenase (LDH) enzyme that was released was evaluated by LDH kit (Biyuntian, China) as the instructions of manufacturer. 50 μL of PRP were used instead of sample suspensions as reference value. The percent of adhered platelets was calculated using the following formula: 𝑝𝑒𝑟𝑐𝑒𝑛𝑡 𝑜𝑓 𝑎𝑑ℎ𝑒𝑟𝑒𝑑 𝑝𝑎𝑡𝑒𝑙𝑒𝑡𝑠 (%) =


× 100%

2.7.6 Plasma Clotting Time The plasma clotting time was adapted from previously reports with some modifications.27 Briefly, 270 μL of normal PPP and 3 mg of samples were incubated at 37 °C. After 3min, 30 μL of CaCl2 (0.2 mol/L) was pipetted, and clotting time was collected, plasma only as control. As comparison, the coagulation factor XII-deficient PPP (Boatman Biotech, China) was also evaluated by the same procedure. 2.7.7 Activated partial thromboplastin time (aPTT) According to previously reports,13, 28 aPTT were assessed by mixing 50 μL of PPP to 50 μL of aPTT reagent (NanJing JianCheng Bioengineering Institute, China). After incubation for 3 min under 37 °C, 50 μL of CaCl2 (25 mM) and the test specimen were added into the test tube at the same time and aPTT was recorded immediately. Moreover, the commercial available Quikclot combat gauze® (QCG) was selected as control group. 2.8 Thrombin Generation

10


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Thrombin–antithrombin complex (TAT), which can be utilized as the indicator of the amount of thrombin generated over a period of time, is a marker of thrombin neutralization.26 Samples were co-cultured with 300 L of heparinized plasma for 10min under 37 °C, then, the thrombin generation in plasma was ceased by adding 20 L of sodium citrate (0.633 M). The levels of TAT were assessed by an ELISA kit (Jonln, China) as the instruction of manufacturer. 2.9 Liver Injury Experiment Liver injury experiment was implemented as previously reports with some changes.1, 16 30 New Zealand White rabbits were anesthetized and randomly divided into 5 groups, then fixed on surgical bench, and the enterocoelia was opened exposing the liver. After that, about 2 cm length and 0.5 cm width of liver organ was cut by surgical scissors, and after10 sec free bleeding, the sterilized samples were applied in the site of defected, and a gauze, GS and CS were also selected as controls for the livers treatment. Following that, hemostasis time and blood loss were record. After 60 min of completion of treatment application, all of the survival rabbits were sacrificed by sodium pentobarbital with overdose. 2.10 Antibacterial Performance Antibacterial activity of specimen for E. coli and S. aureus was assessed as previously reports with some changes.29 Briefly, 10 mg of specimen were put into a 48well plate and sterilized under UV irradiation. After 2 h,10 L of bacterial suspension (~1107 CUF mL-1) was dropped onto each specimen, incubated for 6 h under 37 °C with 90 % humidity inside of microplate. Subsequently, any bacterial survivors were resuspended by adding 1 mL of sterilized PBS into each well, and 1 mL of PBS with 10 L of bacterial suspension was used as control. After that, 10 L of resuspension was dropped onto the agar-plate co-cultured for 18-24 h under 37 °C to observed the surviving bacterial. Furthermore, 100 μL of the resuspension was spread onto plates after serials of 10-fold attenuation in sterilized PBS, and counted the number of colonyforming on the plate. Each group was repeated three times and the results were calculated as kill %: 𝐶𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 ― 𝐶𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑠𝑎𝑚𝑝𝑙𝑒 𝑘𝑖𝑙𝑙(%) = × 100% 𝐶𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡 𝑜𝑓 𝑐𝑜𝑛𝑡𝑟𝑜𝑙 2.11 In Vivo Wound Healing According previous reports,

17, 30

full-thickness infected skin wound healing 11



experiments were carried out with minor revision. SD rats (weight of 200–250 g) were anesthetized with sodium pentobarbital by an enterocoelia injection (40 mg kg−1). Then, four 10 mm diameter circular full-thickness skin traumas on the back were fabricated and coped with 50 μL bacteria suspension (E. coli and S. aureus blend suspension, 1×108 ∼ 1×109 CFU mL−1) and 10 mg of samples. Furthermore, with 10 mm diameter circular uninfected and infected injuries were used as negative control and positive control, respectively. The injuries area was photographed on day 1, 5, 10, and 15 postoperation. After that, the wound region was analyzed by the software of ImageJ (NIH, USA), and the closure rate of wound was evaluated as the following formula: 𝑐𝑙𝑜𝑠𝑢𝑟𝑒 𝑟𝑎𝑡𝑒 (%) =

𝐴0 ― 𝐴𝑡 𝐴0

× 100%

where A0 and At are the wound area at initial state and the set time t.31 The rats were sacrificed on days of 5, 10 and 15, and the tissues of wound region post-operation were collected and fixed with 4% paraformaldehyde solution. Hematoxylin and eosin (H & E) staining were adopted to analyze the pathological section of the tissues of wound region. And a microscope (NIKON, Japan) was used to obtain histological images of wound tissues. All animal operation were in accord with the NIH guidelines (NIH Publication No. 85e23 Rev. 1985) as approved by Shanghai University of Traditional Chinese Medicine.

2.12 Statistical Analysis All the experimental results are expressed as the mean ± standard deviation. The differences were considered to be statistically significant when the p values were less than 0.05.

3 Results and Discussion 3.1 Synthesis and Characterization of MCF, and the Composite Hydrogel Sponges Inspired by the previous reports, MCF, with open large-size pore and more direct interaction with proteins,32 was selected as the initiator for coagulation factors and first synthesized. Compared with the traditional MS (Figure S1), MCF showed a typical spherical-like morphology and uniform pore-covered surface (Figure 1A & 12


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B). Furthermore, TEM and N2 absorption-desorption revealed a continuous ultra-large spherical pore system and large pore size (18.4 nm) (Figure 1C). As anticipated, MCF exhibited significantly more enhanced procoagulant activity than that of the traditional MS (Figure S2). Prior to preparation of the composite hydrogels, the MATMA grafted HEC (quaternized HEC, QHEC) was first prepared by radical grafting method. The appearance of spectra at 1458 cm−1 corresponding to the QA group17 and the enhancement of peaks at 2918 and 1064 cm−1 assigned to the saturated C-H and ether C-O in FTIR (Figure S3A), together with 1H signals at  1.0 and 3.1 ppm assigned to the CH3-C and CH3-N+ groups33 in 1H NMR (Figure S3B) confirmed a successful conjunction of MATMA to HEC. It is worth mentioning that the 1H signals of the glucose units of HEC in the range of  3.23– 3.74 ppm in the 1H NMR were decreased after MATMA grafted, which might relate to the oxidative degradation of HEC. To avoid this undesired oxidative degradation, the composite hydrogels afterwards were prepared by utilizing one-pot method. Specifically, the all precursors were first mixed homogeneously, and then were activated by APS and crosslinked by PEGDA to form QHM composite hydrogel. The QHM with the MATMA/HEC (w/w) ratio less than 2.486 exhibited slight cytotoxicity (Figure S4), and therefore, it was chosen for the following hydrogel preparation and named QHM0. Rheological behavior of the QHM0 hydrogel (Figure 1D & E) revealed a low storage modulus (G’) and viscosity (𝜂’) at the initial 45 s, followed with an abrupt increase as the cross-link forming, indicating a typical sol-gel transition in the process. Finally, the G’ of QHM0 is much larger than 1 times of loss modulus (G’’), demonstrating a formation of true gel.34 In

13C

NMR

spectrum (Figure 1F), the 13C signals at  77.5 and 98.1 ppm assigned to the glucose units of HEC, 13C signals at 55.9 ppm corresponded to the -N-(CH3)3 group and the 13C signals at 33.6 ppm belonged to the crosslinker of PEGDA demonstrated a successful synthesis of QHM0. To endow QHM with the properties of coagulation factors activation and achieve an optimal synergistic effect, various amount of MCF were introduced into the hydrogels, and their effect on water absorption and coagulation factors activation were first characterized (Figure S5) in advance. The results indicated that at low ratio (0.05/0.5), MCF was insufficient to facilitate coagulation factors activation, but exerted significantly negative influence on water absorption of the QHM at high ratio (0.5/0.5). Therefore, the ratio of 0.15/0.5 and 0.3/0.5 , named QHM1 and QHM2 (as listed in Table1) were selected for the following study. The rheological properties of the QHM 13



hydrogels with various MCF content in Figure 1G & H indicated an enhanced G’ and 𝜂’ with the increasing of MCF. Especially, QHM2 exhibited rapider increasing of G’ and 𝜂’ than that of QHM1and QHM0. This phenomenon should be related to the adsorption of MATMA, PEGDA and HEC on the surface or pore of MCF by hydrogen bond and partial chain growth and cross-link generated on the surface or pore of MCF during the reaction.35 Furthermore, the FTIR characteristic peaks of MCF (Si-O-Si peak at 803 cm−1,Si-O-H peak at 973 cm-1 ) and QHM0 (enhanced C=O peak at 1732 cm-1 of PEGDA) 11, 24, 36 are observed in both of QHM1 and QHM2 (Figure 1I). Additionally, the results of TG showed that QHM0 was thermal-degraded completely, and the contents of MCF in QHM1 and QHM2 were 9.8 w/w % and 18.6 w/w % (Figure 1J). Moreover, the rheological behavior of QHMs in its swollen state under 37 °C suggested that the adequately cross-linked state and elastic properties, and the mechanical behaviors of QHMs were improved with the introduction of MCF (Figure 1K).

14


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Figure 1. Synthesis and characterization of the MCF and QHMs. (A and B) The SEM morphology of MCF under different magnification. The MCF showed a typical spherical-like morphology and uniform pore was observed on the surface of MCF (red scale bar: 2 µm and white scale bar: 100 nm). (C) TEM image of MCF, confirming a continuous ultra-large spherical pore in MCF. Insert of C was pore-size distribution of MCF characterized by N2 adsorption/desorption. (D) Rheological behavior of the QHM0 hydrogels at strain of 1%. (E) The oscillatory shear rheology of QHM0 hydrogels at strain of 1%. (F) The NMR spectrum of the as-prepared QHM0. (G) Rheological behavior of QHMs hydrogels at strain of 1%. (H) The oscillatory shear rheology of QHM hydrogels at strain of 1%. (I) FTIR spectra of the asprepared QHMs hydrogels. (J) TG characterization of QHM0, QHM1, QHM2 and MCF. (K) Rheological behavior of QHMs hydrogels at strain of 0.1%.

Porous structure is crucial for a hemostasis material to enlarge the surface area for blood components adsorption.25, 37 The original morphologies of QHMs displayed a similar bulk state after lyophilizing (Figure 2A). Moreover, the surface structure was observed by SEM at different magnification (Figure 2B). QHM0 showed a hierarchical porous structure at 50- and 200-fold amplification and smooth surface in 50000-fold magnification. Comparatively, QHM1 and QHM2 exhibited a similar hierarchical porous structure. Specifically, there clearly existed MCF particles in the surface of QHM1 and QHM2. Furthermore, the MCF loaded in QHM1 and QHM2 exposed clearly pore structure, which will be beneficial for plasma protein contaction.

15



Figure 2. Morphologies of the composite hydrogel sponges. (A) Original morphologies of the composite hydrogel sponges. (B) Morphologies of the composite hydrogel sponges observed by SEM at different magnifications. The images at 50- and 200-fold amplification showed that QHMs posed hierarchical porous structure, and the surface of QHMs, with the increasing of MCF loading, was transferred from smoothness to roughness and almost covered by MCF. Additionally, the images at 50000-fold amplification revealed that the MCF loaded in QHMs exposed clearly pore structure.

3.3 Injectability and Water-triggered Swelling Rapid and efficiency water absorption as well as swelling properties would endow hemostatic materials to promote blood components concentration, fill wound area, stress blood vessels and form physical barrier to cease hemorrhage, especially easy delivery into these deep, penetrating and difficult to access wound in compressed state.1, 16


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7, 11-12

As schemed in Figure 3A, the QHMs could be compressed and injected into the

trauma cavity. Once encountered water, the compressed QHMs with interconnected and macroporous structures expanded immediately, and eventually full-fill into cavity. Figure 3B demonstrated that QHM1 posed a third of original volume after compression and could easily be injected. Also, the water-triggered volume expansion property, due to the existence of charged and hydrophilic group, led the compressed QHM1 to immediately expand and to fully fill into the vessel when encountered water (Figure 3C). Furthermore, the microtopography alteration of QHMs (Figure 3F) revealed that all the hydrogels presented collapsed, almost closed pores structure in the compressed state. After water-absorbing, all the microtopography of QHMs were similar to their original state. These unique properties endowed QHM a potential application for these too deep to access bleeding area and irregular wound to cease hemorrhage. The specific expansion process of QHM1 in PBS is presented in Figure 3D, without any disturbance, QHM1 could rapidly contact, absorb water and expand immediately. As comparison, commercial available GS almost did not absorb any water within 20 sec. Furthermore, the water absorption capacity of the prepared QHMs were compared with DI, PBS (pH = 7.4) and PPP in Figure 3E. As shown, QHM0 had the highest swelling ratio of 7013.4 ± 310.4% by mass at equilibrium, and the swelling ratios of QHM1 and QHM2 gradually decreased to 5780.4 ± 538.9% and 4126.7 ± 411.8%. Similarly, the equilibrium swelling percentages of QHM0, QHM1 and QHM2 exhibited the same trend in PBS and plasma. Comparatively, the equilibrium swelling percentages in water was higher than that of percentages in PBS and PPP, which should relate to the possible charge shielding and osmotic pressure differences due to the buffering ability and ionic strength of both PPP and PBS.7,

23

The superabsorbent

properties should be beneficial for the ensuing blood components concentration. This study indicated that the MCF introduction exerted negative on the water adsorption capacity, especially at higher MCF content.

17



Figure 3. Swelling and shape recovery property of the QHMs hydrogel sponges. (A) Schematic drawing of the injectability and water-triggered instant volume expansion for QHM. When the compressed sample encountered water, the volume of sample was expanded immediately. (B) The compression properties and injectability of QHM1. (C) Water-triggered swelling properties of QHM1. (D) Digital images of swelling processes of QHM1 and gelatin sponge in PBS. (E) The corresponding swelling ratios of samples in DI, PBS and PPP over 10 min. (F) Morphologies of the composite hydrogel sponges in compressed state and shape recovery state. All the hydrogel sponges presented collapsed, almost closed pores structure in the compressed

18


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state. After water adsorption, all the hydrogel sponges showed similar in morphologies to their original state. (**, p< 0.01; ***, p< 0.001)

3.4 Cytotoxicity and Hemolysis of the QHM Hydrogel Sponges Nontoxic or minimally toxic to the body is what an ideal biomaterial should have. Therefore, the cytotoxicity of various samples on C2C12 cells was evaluated by MTT and Live/Dead staining. Figure 4A showed that viabilities of the cells, after co-cultured with all samples, were greater than 95% of that of in normal media growth cells. Also, a live/dead cell viability assay with green fluorescence (Figure 4B) demonstrated a superior cytocompatibility of the samples. Hemolysis is another crucial index for assessing the biocompatibility of hemostatic. According to previously reports, cation polymers may possess hemolytic side effects.14, 38

The hemolysis ratio with concentrations ranging from 2 mg to 10 mg mL-1 exhibited

an insignificantly hemolytic activity (lower than 5%) (Figure 4C), demonstrated much better biocompatibility for RBC. Moreover, most of the RBCs were attached on the surface of samples after hemolysis test (Figure 4D).

Figure 4. Cytotoxicity and Hemolysis of the QHMs. (A) Cell viability of QHMs on C2C12 by 19



MTT. Cells viability all were greater than 95% of that in normal medium. (B) Images of C2C12 cells after co-culture with samples. All of cells were stained with green fluorescence, demonstrating lower cytotoxicity. (C) Hemolysis assay for various concentration samples at 37 °C for 1 h. QHMs all exhibited lower hemolytic activity and great biocompatibility for RBC. (D) Images of RBCs incubated with samples for 1h under 37 °C. Most of the RBCs were adhered on the surface of samples. (scale bar: 100 m)

3.5 In Vitro Hemostasis Performance of the QHM Hydrogel Sponges The blood-clotting capability of the QHMs were first evaluated in vitro by BCI, in which a slower clotting rate reveals a higher absorbance value of the hemoglobin solution.1,

25

As shown in Figure 5A, the BCI value of GS (68.04  6.73 %) was

significantly higher than that of QHM0 (5.27  3.41 %), QHM1 (5.51  1.67 %) and QHM2 (5.62  3.79 %), respectively, meaning a better clotting ability of QHMs. Also, Figure 5B showed the images of the blood clotting index process. It can be seen that QHM groups all exhibited excellent blood contact efficiency, and almost no RBCs were diffused from QHM groups into DI, and the color of rinsing DI in QHM groups slightly changed even after shaking, indicating blood could be automatically coagulated on the QHM groups within 5 min. Whereas little clotted blood was found in blood only and GS group. Next, the hemostatic mechanism of the hydrogel sponge was further explored. It is well-accepted that hemagglutination and platelet aggregation play dominant in the primary hemostasis.25 Therefore, the surface attachment and morphologies of RBCs and platelets were observed. GS, a commercial hemostatic product to induce hemostasis via typical blood cell aggregation, was used as control groups. The RBCs and platelet attachment were tested and indicated that (Figure 5C & E) QHM1 exhibited the best cells adsorption ability for RBC and platelet than other groups. Also, as shown in Figure 5D & F, all of QHMs showed a great number of RBCs and platelets adhering than that of GS. Moreover, all the QHMs hydrogel sponges exhibited many activated platelets aggregation with changed shape from irregular distinctive disks to circular deformation.1 Furthermore, by H&E staining, it can be confirmed that the blood cells were trapped into the pores of QHMs, which can promote blood cells aggregation (Figure 5G). The hydrophily and superabsorbent properties of QHMs, which could induce the blood cells into its network and concentration,1 together with the electrostatic interaction between QA and blood cells18, 39 could initiate hemagglutination and platelet aggregation (Figure 5H) as well as facilitate coagulation. 20


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Figure 5. In vitro hemostatic capacity evaluation of QHMs. (A) Corresponding blood clotting index of various QHMs. (B) Photographs of the blood clotting index process. The QHMs displayed a higher blood coagulation speed and a lower BCI than GS group. (C) 21



Corresponding RBC absorption on various QHMs. (D) SEM photos of RBC attachment on the surface QHMs and GS. (E) The platelet absorption on different materials. (F) SEM photos of platelet attachment on the surface of QHMs and GS. (G) H&E staining of blood clots. It revealed that the blood cells were trapped into the pores of QHMs, which can promote blood cells aggregation. (H) Schematic illustration of the aggregation of the blood components adhered on the composite hydrogels by water-triggered concentration and electrostatic interaction. (white scale bar: 100 µm. red scale bar：20 µm. *, p< 0.05; **, p< 0.01)

Coagulation factors activation is another key element for the secondary hemostasis.13 As previously reports, silica-based material can promote blood coagulation by contacting activation of FXII.40 To determine the function of MCF played in the activation of FXII, the clotting time in normal plasma and FXII deficient plasma of the QHMs were compared. It can be found that in the normal plasma, QHM1 (59  4 %) and QHM2 (84  4 %) showed shorter clotting time than that of QHM0 (112  10 %), but there were not different in the FXII deficient plasma (Figure 6B). In addition, the PT and aPTT of the MCF-treated plasma were decreased dramatically (Figure S2). All of these decreasing may be caused by initiating the intrinsic pathway,16,

36

and then stimulate the thrombin forming in the common

coagulation pathway (Figure 6A). In order to confirm this hypothesis, the formed thrombin-antithrombin complex was detected. The results demonstrated that QHM1(91.89  14.38 ng/mL) and QHM2 (62.98  7.34 ng/mL) had substantially higher TAT levels than that of QHM0 (55.33  9.05 ng/mL) and control group (51.22  11.06 ng/mL) (Figure 6C), which implicated that the incorporation of MCF indeed led to a reduced clotting time by coagulation factors activation. It is worth to note that, consistent with the trends observed at RBC and platelet adhesion, a higher MCF loading led to the reduction of TAT level. This phenomenon should be related to the specific interaction between QHMs and FXII. A higher surface covering of QHMs by MCF could mask more of QA group as the increasing of MCF, and thus weaken the interaction between QHMs and FXII.18, 37, 39 Moreover, the secondary coagulation time were tested by aPTT. QCG, a commercial product to achieve hemostasis by secondary coagulation activation, was chosen as control group. The results demonstrated that QHM1 and QHM2 exhibited a similar procoagulation properties as QCG (Figure 6D). From above results, it can be confirmed that appropriate ratio of MCF incorporation can promote FXII transformation and then accelerate thrombin forming, and eventually facilitate hemostasis.

22


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Figure 6. Coagulation factors activation of QHMs hydrogel sponges. (A) Process of the coagulation cascade, which includes intrinsic and extrinsic coagulation pathways (including the common pathway). (B) Coagulation time of normal plasma and FXII deficient plasma of QHMs hydrogel sponges. Compared to the coagulation time of normal plasma and FXII deficient plasma, it can be inferred that the MCF loaded in QHMs could active coagulation FXII to promote clotting formation. (C) Thrombin–antithrombin complex of thrombin generation over time. (D) Corresponding aPTT results of the QHMs hydrogel sponges compared to QCG. (*, p< 0.05; **, p< 0.01)

3.7 In Vivo Hemostatic Behavior Hemostatic performance of QHMs were further assessed by detecting the blood loss and hemostasis time both in the non-compressive rat-tail amputation (Figure S5) and rabbit-liver lethal defect model (Figure 7). The commercial available GS and CS were employed as control group. For the mouse-tail amputation, QHM0, QHM1 and QHM2 groups presented significantly lower blood loss and shorter hemostatic time than that of blank group, CS and GS group (Figure S5 B & C). And there was no significant difference among QHM0, QHM1 and QHM2. We think this phenomenon may be related to the small wound area and less blood loss of rat-tail amputation. A 23



QHM-based fast blood absorption could fully function to cease bleeding and achieve a desirable hemostasis, and the endogenous coagulation function of MCF played negligible effect and thus there is no obvious superiority of QHM1 and QHM2 groups over the QHM0. Next, with rabbit-liver lethal defect model, QHM0 and QHM1 were selected for further hemostasis evolution. As shown in Figure 7E, a complete hemostasis of QHM1 was achieved in 45  8 s, compared to 584  30 s in control group, 70  10 s in CS group, 130  13 s in GS group and 75  7 s in QHM0 group. Moreover, QHM1 showed a less blood loss than other groups (Figure 7C). Additionally, all rabbits in the group of CS, QHM0 and QHM1 were survival, one of six in GS group was died， and half in control group were survival. This obvious superiority of QHM1 over the QHM0 confirmed, besides the water adsorption and protein concentration capacity, an important role of the MCF-induced endogenous coagulation.

Figure 7. Hemostatic behavior of lethal liver defect models. (A) Photographs of the lethal liver defect models after hemostasis. (B & C) Hemostasis time and blood loss of the lethal liver defect models. (** p< 0.01, ***, p< 0.001).

3.6 In Vitro Antibacterial Wound infection is another challenge that delay healing or even nonhealing wounds.16 E. coli and S. aureus were employed to evaluate the anti-microbico activity of samples. As shown in Figure 8C & D, the imaged survival bacterial demonstrated that all the QHMs had superior antibacterial properties for both E. coli and S. aureus. Quantitative analysis displayed that the bactericidal ratios of E. coli and S. aureus on 24


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QHMs were more than 99 % (Figure 8B). Furthermore, both E. coli and S. aureus presented a roughness surface and deformed cellular structure after contacting with the surface of QHMs (Figure 8E). These results confirmed that the as-prepared samples possess great antibacterial ability. The QA group, as previously reported, can disrupt the negatively bacterial membranes, and further cause bacterial death (Figure 8A).17, 38 Moreover, due to the different structure of cell membrane between mammalian cells and bacterial, the cationic hydrogel with low QA density exhibited excellent cytocompatibility.17, 38 Furthermore, similar with cationic antibacterial peptides, QHM should pose a low tendency for the development of bacteria resistance.38

25



Figure 8. In vitro antibacterial activity of QHMs. (A) Schematism of the antimicrobial mechanisms of the QHMs. Bioinspired with antibacterial peptides, QA group on the QHM can disrupt the membrane of bacterial, leading to bacterial death. (B) Quantitative antibacterial activity of various QHMs and QCG for E. coli and S. aureus. (C & D) Survival bacteria colonies images of E. coli and S. aureus on agar plates after treated with samples. (E) SEM photos of E. coli and S. aureus after co-cultured with QHMs and QCG for 2h. Red arrows indicate morphology change, and lesions of pathogens. (scale bar: 200 nm.)

3.8 Wound Healing Nowadays, wound healing presents a significant challenging worldwide. Normally, healing of wound includes four phases-hemostasis, inflammation, proliferation, and tissue remodeling. Immediate and effective hemostasis and antibacterial could provide a suitable micro-environment to keep the moisture, reduce pathogens aggression and inflammatory response of the wound site and thereby facilitated the wound healing (Figure 9A).1, 29, 41 Therefore, the potential application of the QHMs on the wound healing was assessed in this research. As presented in Figure 9B, the images of the bacterial infected full-skin defect wound-healing process were recorded at set time points that the defects treated with QHM1and QHM2 were smaller than other groups after 5 days, and fully healed on day 15. Furthermore, the quantitatively wound-close ratio was collected (Figure 9C), indicating that the healing ratios of QHM1(62.8 %) and QHM2 (58.2%) were faster than that of uninfected group (54.4%), infected group (42.2%) and QHM0 treated group (52.3%) after 5 days treatment. These results demonstrated that QHMs possessed a potential to accelerate wound healing. H&E staining was utilized for wound histomorphological characterization (Figure 9D). It revealed that the uninfected wounds existed a substantially higher level of inflammatory cells than the infected wounds. After QHMs treatment, the number of inflammatory cells significantly decreased, especially in QHM1- and QHM2-treated groups on day 5 and 10, which demonstrated that the synthesized QHM could effectively inhibit wound infection. Moreover, at day 5, QHM1- and QHM2-treated groups showed a dense dermis tissue under scab; and a newly complete epidermis layer was formed at day 10. This QHMs-induced promote wound healing should be related two respects: (i) QHMs with rapid hemostasis performance could provide a desirable micro-environment to promote cells proliferation of wound area;3 (ii) QHMs could avoid bacteria infection and decrease the level of inflammation response in the early stages of wound healing.42 Above all, it revealed that QHM1 and QHM2 could promote 26


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wound healing process and exhibited immense probability as wound dressings.

Figure 9. Wound healing performance of the QHMs in vivo. (A) Schematic illustration of the QCMs-facilitated wound healing. The QHMs-induced rapid hemostasis and antibacterial could provide a desirable micro-environment, and which facilitated skin healing. (B) Wound images on special day of post-operation. (C) Closure rate of various samples treated wound on special day of post-operation (*, p < 0.05; **, p < 0.01). (D) Results of wound tissue staining by H&E (scale bar: 200 μm；S, scab; E, epidermis; I, inflammation).

27



In today’s clinic and battlefield urgent treatment, rapid hemorrhage controlling and effective antibacterial is still a great challenge. The QHM composited hydrogel sponge developed here exhibited much better hemostasis performance than the commercial CS and GS. According the previous reports and this research results, it could be deduced that the main contributor of QHMs was mainly attributed to the QHMs-triggered faster and massive water absorption ability, the MCF-derived rough surface and partially QA group exposure, which led to a rapid blood components concentration, enhanced RBC adhesion and platelet attachment, and thus promoted the formation of a primary platelet plug and hemagglutination.1,

14

On the other hand, appropriate amount of MCF

introduction (9.8%) not only exerted negligible influence on the protein absorption, but also facilitated the activation of blood coagulation factors, which accelerated the strong fibrin gel formation to prevent blood loss.13 As for the excellent antibacterial property and prominent cyto-compatible, it should be related to the appropriate QA group in the hydrogel sponge. In addition, QHM1 exhibited an anti-infection ability and could facilitate wound healing in the full-thickness skin wound models. Furthermore, from the standpoint of convenience of clinic use, the QHM is easy for delivery to these deep, penetrating and difficult to access wound by an injector or other devices in compression state. Another respect, for practical preparation and costs, QHM manufacturing process is simple, without huge energy and expensive chemicals consumption. Therefore, this research represents an advancement step in the exploitation of hemostasis material with enormous commercial and clinical possibility.

4 Conclusions In this study, a bioinspired, quaternized HEC/MCF composite hydrogel sponge was successfully designed and synthesized via radical graft copolymerization. The asprepared QHM (amount of MCF 9.8%) with excellent water-triggered swelling property and superabsorbent capacity could not only present efficient excellent hemostatic effects in vitro and in vivo, but also displayed desirable antibacterial ability. More importantly, the effective hemostatic was achieved more successfully than commercial gelatin sponge and chitosan powder hemostatic. Additionally, QHM, due to the immediate and effective hemostasis as well as antibacterial capacity, could maintain a suitable moisture micro-environment to reduce pathogens aggression and inflammatory response of the wound site and thereby facilitated the wound healing. Furthermore, QHM showed excellent biocompatibility and negligible hemolysis. We believe this study gives an alternative method for deep and noncompressible bleeding 28


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control.

Acknowledgements This research was jointly supported by the National Natural Science Foundation of China for Innovative Research Groups (No. 51621002), Joint project of Ministry of Education (6141A02022628), Science and Technology Commission Shanghai Municipality (16441902800). Also, the authors gratefully acknowledge the and Leading Talents in Shanghai in 2017.

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Table of contents

Inspired by the coordinated primarily formation of stable clot and secondly coagulation activation in natural hemostasis process, an injectable, quaternized hydroxyethyl cellulose (HEC)/mesocellular foam (MCF) composite hydrogel (QHM) for both hemorrhage control and effective antibacterial were designed and prepared via one-pot radical graft copolymerization. The QHM with rapid water-triggered expansion, 32


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platelet plug, hemagglutination and MCF-initiated coagulation factors activation properties exhibited efficient hemostatic effects in nonocompressible hemorrhage model. Furthermore, the QHM with QA groups density of 2.732 mmol/g exhibited remarkable antibacterial activities and excellent cytocompatibility, thus provide suitable micro-environment to enhance wound healing.

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Bioinspired, Injectable, Quaternized Hydroxyethyl Cellulose

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