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Characterization, Synthesis, and Modifications

Synthesizing Functional Biomacromolecular Wet Adhesive with Typical Gel-Sol Transition and Shear-Thinning Features Luyao Gao, Shuanhong Ma, Jiajun Luo, Guangjie Bao, Yang Wu, Feng Zhou, and Yong-Min Liang ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/ acsbiomaterials.9b00939 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 4, 2019

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ACS Biomaterials Science & Engineering

ACS Biomaterials Science & Engineering Article type: (Characterization, Synthesis, and Modification) Synthesizing Functional Biomacromolecular Wet Adhesive with Typical Gel-Sol Transition and Shear-Thinning Features Luyao Gao 1,2, Shuanhong Ma 2*, Jiajun Luo 3, Guangjie Bao 4, Yang Wu2, Feng Zhou2*, Yongmin Liang1* 1State

Key Laboratory of Applied Organic Chemistry, Lanzhou University, Lanzhou 730000, P. R. China 2State Key Laboratory of Solid Lubrication, Lanzhou Institute of Chemical Physics Chinese Academy of Sciences, 18 Middle Tianshui Road, Lanzhou 730000, P. R. China 3Institute of Orthopaedic & Musculoskeletal Science, Division of Surgery & Interventional Science, University College London, Royal National Orthopaedic Hospital, Stanmore, HA7 4LP, United Kingdom 4 College of Dentistry, Northwest Minzu University, 1 New Northwest Villiage, Lanzhou 730030, P. R. China. Corresponding Authors: *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Abstract: Functional wet adhesives become increasely important in the field of biomedicine. Herein, modified chitosan (CS) biomacromolecule is synthesized as tissue wet adhesives and haemostatic materials. In typical case, after getting through two steps of organic reactions, catechol and lysine groups are grafted onto the chitosan backbone, enables to successful preparation of one kind of novel biomacromolecule of chitosan-catechol-lysine (CHIC-Lys). The as-prepared CHIC-Lys biomacromolecule shows improved wet adhesion strength, concentration-dependent gel-sol transition feature when shear stress is cycled between low stress (0.2 Pa) and high stress (2 Pa) at room temperature and obvious shear-thinning feature under wide concentrations range, along with good biocompatibility, comparable with traditional CS. Based on these obvious characteristics, CHIC-Lys is successfully coated onto the surface of syringe needle, and the decorated needle shows considerable haemostatic effect in test of rats venous bleeding. Overall, the as-synthesized CHIC-Lys biomacromolecule exhibits considerable application potential in biomedical field. KEYWORDS: biomacromolecule, wet adhesion, sol-gel transition, shearing-thinning, hemostasis

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1. Introduction How to effectively arrest bleeding after hemorrhage has been plaguing us. Two-thirds of the failures in surgery are caused by failure to stop bleeding in time. Failure to arrest bleeding in a timely and effectively manner is the main cause of mortality.1 With the advancement of society and the development of science and technology, many non-absorbable haemostatic materials have been used clinically.2-3 Although it has played an important role in hemostasis, problems such as wound infection in the later stage still plague us.4-7 Therefore, research and development of an effective absorbable haemostatic material has become a concern for us. In the field of biomedical engineering, many materials have good adhesion properties, such as alginate systems8 and glycosaminoglycan-based complexes9. More and more people are favoring chitosan.10 Chitosan (CS) is easily dissolved in weak acid solvent, and it is particularly worth nothing that the dissolved solution contains an amino group (NH2+) which inhibits bacteria by binding negative electrons.11-12 Chitosan inhibits bacterial activity, making it widely used in medicine, textiles and food.13-15 Besides, chitosan has the function of promoting blood coagulation, and it can be used as a haemostatic agent.16 It can also be used as a wound filler material, which has the functions of sterilization, promoting wound healing, absorbing wound exudates, and not easily dehydrating and contracting.17-20 A chitosan haemostatic dressing has been reported which has good haemostatic and antibacterial effects.21-22 However, due to the strong hydrogen bonding between the intramolecular and intermolecular of chitosan, it is difficult to dissolve in neutral or alkaline water.23 The ability to inhibit bacteria of chitosan is only in acidic medium. So the application range of single chitosan is narrow. Based on this problem, Kwon et al. used an amide bond to


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covalently attach a hydrophilic sugar moiety, gluconic acid to prepare chitosan derivatives to broaden the water solubility in a wide pH range.24 Although the result was indeed that its solubility was improved. However, increasing its solubility reduced its wet adhesion characteristics. Consequently, a series of means were employed to improve the wet adhesion characteristics of chitosan. For example, Lee et al. improved the wet adhesion of chitosan by introducing dopamine groups onto it backbone, resulting in enhanced wet adhesion,25 based on the strong covalent and non-covalent interaction of phenolic hydroxyls with various surfaces.26-27 The as-synthesized biomacromolecules could be assembled onto a needle for successful hemostasis. Nevertheless, synthesis conditions for such biomacromolecule were harsh and required precise control of the oxidative cross linking of dopamine. In addition, since the molecule had strong wet adhesion without shear-thinning characteristic, it can be imaged that the rising friction against the wet tissue when the needle was inserted into or removed out is bound to be large, causing pain to the patient. So the question is that can be synthesize functional chitosan-based wet adhesion with considerable solubility, adhesion strength and shear-thinning feature? In this study, we attempted to develop one kind of novel biomacromolecular wet adhesive with typical shear-thinning properties that can may be used to stop bleeding. We selected chitosan as the central framework to synthesize wet adhesive because of its good biocompatibility, blood compatibility and biodegradability. First, catechol and L-lysine (Lys) groups were continuously grafted on the side chain of chitosan after two amide reactions to allow the preparation of catechol/Lys-conjugated chitosan (CHIC-Lys) functional biomacromolecule. As expected, the catechol groups in CHIC-Lys can exhibit considerable



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wet adhesion, while the existed Lys groups can enhance the water solubility of the CS skeleton

along

with

improved

antibacterial

properties.28-29

Subsequently,

relative

physical-chemical characterizations, such as Fourier Transform Infrared Spectroscopy (FT-IR), Gel Permeation Chromatography (GPC) and 1H NMR, were empolyed to prove the successful synthesis of CHIC-Lys. Furthermore, wet adhesive, rheological and frictional properties of CHIC-Lys were measured systematically. Finally, the hemostatic effect and the biocompatibility of CHIC-Lys were evaluated.

Figure 1│Synthesis and structure of CHIC and CHIC-Lys.

2. Experimental Section. 2.1. Materials. Chitosan (CS, deacetylation rate: 85%) and L-Lysine were purchased from

the

Chemical

Reagent

Co.

of

J&K

Chemical

Ltd.

(Beijing,China).

3-(3,4-Dihydroxyphenyl)propionic acid (HCA, 98%) was purchased from Energy Chemical (Shanghai,China). N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and N-Hydroxysuccinimide (NHS) were purchased from J&K Chemical Ltd. All of above reagents were commercially available and used without any purification. All other chemicals


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were of analytical grade. Deionized water was applied for all polymerization and treatment processes. 2.2. Preparation of Catechol-Conjugated Chitosan CHIC was prepared as previous literature reported.30 Briefly, chitosan (0.5 g) was dissolved in deionized and distilled water (DDW, 50 mL). Besides, 1 % (v/v) glacial acetic acid was added dropwise into chitosan solution and stirred vigorously. While the solution became clear and transparent, hydrocaffeic acid (591.0 mg) and EDC (1244.9 mg) in 25 mL of DDW and ethanol (1:1 v/v) were added to the chitosan solution, and reacted for 12 h at room temperature. Ethanol was removed by rotary evaporation. The product was purified by dialysis (MWCO: 8000-14000, SpectraPor) against pH 5.0 HCl solution in DDW for 2 days and DDW for 4 h. The final product was freeze-dried. 2.3. Preparation of Catechol-Conjugated Chitosan grafted lysine CHIC-Lys was prepared by conjugating lysine onto the amino groups of CHIC. In brief, CHIC (300 mg) was dissolved in 25 mL DDW, the mixture which included lysine (150 mg), EDC (196 mg) and NHS (118 mg) was added into above mentioned solution, reacted for 24 h at room temperature. The product was purified by dialysis (MWCO: 8000-14000, SpectraPor) against DDW for 2 days. After lyophilization, the white sponge solid could be obtained. 2.4. Characterizations. 2.4.1 Physical-chemical properties characterizations FT-IR spectrum was obtained on a Perkin-Elmer Transform Infrared Spectrometer (Perkin-Elmer). The polymer compositions were further determined by 1H NMR (400 MHz) analysis using 400 MHz spectrometer (Bruker AM-400) in Deuterium oxide (D2O). The



molecular weights of the biomacromolecule were determined by a gel permeation chromatography (GPC) analyzer (concentration: 5mg·mL-1, pure water as eluent). Preparing a series of gradient concentrations of the CHIC-Lys solutions (0.001 mgmL-1~10 mgmL-1), titanium-coated silicon wafers immersed in the different concentrations of the CHIC-Lys solutions for 12 h. A series of silicon wafers prepared were used for atomic force microscopy (AFM) tests in air. Atomic force microscopy morphologies were measured by an Agilent Technologies 5500 AFM using a Mac Mode Pico SPM magnetically driven dynamic force microscope in tapping model. The sessile water droplet contact angle (CA) measurement was conducted using a DSA-100 optical contact angles meter (Kruss, Germany) at 25 ℃ . The Quartz crystal microbalance with dissipation (QCM-D) measurements was performed to take adsorption assay 25 ℃ by a Q-Sense microbalance (Sweden) and used the commercial gold-coated quartz chips (QSX-301, Q-Sense). 2.4.2 Adhesive, rheological and frictional properties characterizations The adhesion properties for polymers were measured by an electrical universal material testing machine (EZ-Test, SHIMADZU) with face-to-face contact mode in air. The schematic diagram of experimental device for the adhesion test was in Figure S5. Specifically, the speed was set to be 10 mm·min-1 in the compress mode to obtain the load-displacement curve. The testing area was kept as 1x1 cm2 against glass substrate. The force applied perpendicular to the plane in all adhesion tests was 5N. After being compressed for 0 s with a load of 5 N, two surfaces were separated and the interfacial adhesion strength can be determined based on the adhesion force curve. We repeated all measurements at least five times and gave the average value.


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The rheological properties of polymer solution were investigated using a rheometer (HAAKE, RS6000, Germany) with a coaxial two parallel plate model where the diameter of the clamp was 35 mm. The contribution of a solid-like behavior (storage modulus G’) and a liquid-like behavior (loss modulus G’’) were recorded with increasing shear stress. The dynamic shear rate and dynamic shear viscosity were detected when the upper platen rotated with an angular velocity at a strain-constant mode. The volume of solution was 1 mL to cover the plate. Frequency was fixed to 1 Hz, as determined using frequency sweep equipment. The friction test was carried on the conventional pin-on-desk reciprocating tribometer (CSM Co. Ltd, Switzerland) by acquiring friction coefficient at 1 N load , frequency = 1 Hz and amplitude = 10 mm. These pins were elastomeric poly (dimethylsiloxane) (PDMS) hemisphere with a diameter of 6 mm. The desks were polished titanium alloy sheets. The lubrication additives were 1 mg·mL-1 CHIC and CHIC-Lys, respectively. Each sample was measured at least 5 times. 2.5. Haemostatic capability of CHIC-Lys in vivo models. All animals experiments were performed in accordance with relative guidelines and regulations of the National Ethics Committee on Animal Welfare of China, and experiments involving animals were approved by the ethics committee at Gansu University of Chinese Medicine. To test in vivo haemostatic properties of CHIC-Lys, we used rats (normal Wistar rats, 200-220 g, 8 w, M) as hemorrhaging model. Specifically, a rat was paralyzed using 10% chloral hydrate solution and immobilized on the board. The bleeding wound site with size of 20.2 cm2 on rat belly. We immediately treated wounds of rats with two kinds of gauze. The first consisted bare gauze without any polymeric coated. The second consisted CHIC-Lys-coated gauze prepared



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by coating with the CHIC-Lys solution after being freeze-dried. The amount of bleeding on the two kinds of gauze within 5 minutes was accurately collected to inspect the arresting bleeding effect of CHIC-Lys. To further evaluate the haemostatic effect in vivo, needles which diameter were 0.7 mm were used to cause bleeding in rats by puncture of the femoral veins. We also prepared two control needles: the first one was bare metal without any polymeric coated, the second one was coated with CHIC-Lys. The amount of bleeding at the injection site was collected and weighted at the same time. All operations followed the ethical protocol provided from the China Ministry of Health and Welfare. All procedures complied with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. All measurements were triplicate. 2.6. Cell Viability Assays. The cell viability was evaluated by CCK-8. 4th generation L-929 cells were seeded into 96-well plates at a density of 8×103cells per well. Incubation was carried out in DMEM containing fetal bovine serum (FBS), 100 unitmL-1 penicillin and 100 gmL-1 streptomycin. CHIC-Lys was completely dissolved in this medium, and incubated at 37 °C for 24 hours. Thereafter, 10 L of CCK-8 reagent was added to each well, and the plate was further incubated at 37°C for 4 hours. Finally, the fluorescence signal was measured by microplate reader (λex = 450 nm). The cell viability was calculated based on the Eq. (1):

𝐂𝐞𝐥𝐥 𝐯𝐢𝐚𝐛𝐢𝐥𝐢𝐭𝐲(%) =

3.

𝐅𝐥𝐮𝐨𝐫𝐞𝐬𝐜𝐞𝐧𝐭 𝐢𝐧𝐭𝐞𝐧𝐬𝐢𝐭𝐲 (𝐬𝐚𝐦𝐩𝐥𝐞) × 𝟏𝟎𝟎 (%) 𝐅𝐥𝐮𝐨𝐫𝐞𝐬𝐜𝐞𝐧𝐭 𝐢𝐧𝐭𝐞𝐧𝐬𝐢𝐭𝐲(𝐜𝐨𝐧𝐭𝐫𝐨𝐥)

Result and Discussion. 8 ACS Paragon Plus Environment

(𝟏)

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CHIC-Lys was synthesized by forming two amide bonds between the carboxylic acid group in hydrocaffeic acid and the primary amine group in the chitosan and the carboxylic acid group of the lysine and the primary amine group of the chitosan, respectively. The chemical components of the polymers were verified by FT-IR spectroscopy, as illustrated in Figure 2A. The peaks appeared at 3250 (-OH), 1625, 1517 cm-1 were characteristic vibration peak of CHIC. The new peak appeared at 720cm-1 which was the C-H in-plane rocking vibration adsorption peak of four or more methylene groups continuously. Its appearance means the lysine successfully grafted to the CHIC. Moreover, the new peak appeared at 3385 cm-1 which was the N-H vibration peak of the secondary amide. The peaks at 1644 cm-1 and 1556 cm-1 were mightily proved the formation of the secondary amide. The synthesized of polymers were further confirmed by using 1H NMR (400 MHz). 1H NMR (400 MHz, Deuterium Oxide) δ 6.86 (s, 3H), 4.46 (s, 14H), 3.74 (d, J = 66.5 Hz 72H), 3.19 (s, 1H), 2.80 (s, 4H), 2.57(s, 2H), 1.98 (s, 10H), 1.34 (d, J = 6.8 Hz, 2H), 1.20 (s, 2H), 1.17 (s, 2H). The peaks which appeared at δ 6.86 (aromtic ring proton) and 3.19 (1H,-CH-NH2) demonstrated the catechol group and lysine successfully grafted to chitosan. Combining the results of IR and NMR, it showed CHIC-Lys was successful preparation. As shown in Figure S1, the number average molecular weight (Mn) of CHIC was 1.254×104, the number average molecular weight (Mn) of CHIC-Lys was 1.540×104. Besides, the Mw/Mn of polymers was 1.332 and 1.312, respectively. The results confirmed the truth of successfully synthesizing the CHIC-Lys. In addition, the Mw/Mn was the heterodisperse index and the results of this experiment showed that the molecular weight distribution of the polymer was relatively uniform.



Figure 2│(A) FT-IR spectra of CHIC and CHIC-Lys. (B) 1H NMR spectra of CHIC. (C) 1H NMR spectra of CHIC-Lys.

Figure 3 │ Atomic force microscope (AFM) images of titanium-coated silicon wafers after assembling different concentrations of CHIC-Lys: (A) 10 mgmL-1, (B) 1 mgmL-1, (C) 0.1 mgmL-1, (D) 0.01 mgmL-1, (E) 0.001 mgmL-1 and (F) bare titanium-coated silicon wafer. The image size was 5 m  5 m. After charactering successful synthesis of the CHIC-Lys biomacromolecule, we firstly studied its assembly, adsorption, wettability and wet adhesion properties. The surface morphology of the titanium-coated silicon wafers after CHIC-Lys adsorption were observed by atomic force microscope (AFM). As shown from Figure 3A to Figure 3E, with increasing of the CHIC-Lys concentration, the surface roughness (Sq, root mean square roughness) of


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the titanium layer increased. No doubt that 10 mgmL-1 CHIC-Lys has the maximum roughness which is 6.94 nm (Figure 3A). By contrast, the surface roughness of the bare titanium sheet was only 1.04 nm (Figure 3F). This indicates that that CHIC-Lys could generate effective adsorption on the surface of the titanium layer by some non-covalent interactions. Especially, the surface roughness of the titanium layer which immersed in 0.001 mgmL-1 CHIC-Lys solution could also change slightly as 1.20 nm, even in very low concentration of CHIC-Lys. Meanwhile, the adsorption of the samples to the substrate could be regarded as microscopic adhesion. The morphology of the sample measured by atomic force microscopy could be used to characterize the adsorption ability of the sample against the substrate from a microscopic point view. The greater the roughness of the wafer was, the more biomacromolecules adhesive on the wafer. Subsequently, contact angles (CAs) of 5 μL water droplet on these assembled surfaces were measured. As shown in Figure 4A, the CA of the bare titanium layer was 95.53°. After assembling CHIC-Lys biomacromolecule with different concentrations, the CAs of titanium layers obviously decreased and were basically stable at 40-50° (Figure 4B-4F). As shown in Figure 4B, a very small amount of CHIC-Lys biomacromolecule adsorbed onto the titanium layer can make the surface more hydrophilic.



Figure 4 │ Digital photographs of water droplets on bare titanium layer (A) and titanium-coated silicon wafers after assembling CHIC-Lys biomacromolecule with different concentrations of (B) 0.001 mgmL-1, (C) 0.01 mgmL-1, (D) 0.1 mgmL-1, (E) 1 mgmL-1 and (F) 10 mgmL-1. Changes of frequency for QCM chip upon injecting different biomacromolecues solution. (G) 1 mgmL-1 CHIC and (H) 1 mgmL-1 CHIC-Lys. Moreover, Quartz Crystal Microbalance (QCM) was employed to quantitatively evaluate the adsorption of CHIC-Lys and CHIC biomacromolecules on the gold chip surface, as illustrated in Figure 4G, 4H. As shown in Figure S6, both of them were rigid adsorption and

followed the Sauerbrey equation. The principle operation of QCM could be described as equation (2): ∆f = ―c∆m

(2)

The crystal oscillation frequency change f is proportional to the mass change m of the deposit on the working electrode, c is a chip-dependent constant.


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As shown in Figure 4G, when 1 mgmL-1 CHIC was pumped into the instrument, the frequency shifted from 0 Hz to -5.8 Hz. While it was washed by water, the frequency shifted from -5.8 Hz to -4.5 Hz. This indicated that CHIC interacted with the gold chip surface by physical adsorption. By contrast, using the same concentration of the CHIC-Lys, the frequency of chip decreased from 0 Hz to -5.6 Hz, while it didn’t change any more upon washing by water (Figure 4H). So CHIC-Lys biomolecule can interact with the gold chip more sufficiently. Above results indicated that CHIC-Lys possessed better adsorption capacity than CHIC.

Figure 5 │ Adhesion strength-displacement curves for CHIC and CHIC-Lys with different concentrations: (A) 0.1 mgmL-1, (B) 1 mgmL-1, (C) 10 mgmL-1. (D) The statistical result of



concentrations-dependent wet adhesion strength of the CHIC and CHIC-Lys against glass substrate. Next, wet adhesion property of the as-synthesized CHIC-Lys biomacromolecule was quantitatively evaluated with considering its potential application as wet adhesive in biomedical field. The adhesion force Vs displacement curves for three different concentrations of CHIC-Lys and CHIC were shown in Figure 5. The experimental results showed that the wet adhesion strength improved with increasing the concentration of CHIC-Lys and CHIC. By comparison, CHIC-Lys with different concentrations exhibited higher adhesion strength than that of CHIC. In a typical case, the adhesion strength 0.1 mgmL-1 CHIC was close to 0, while it could achieve to ~ 20 KPa for 0.1 mgmL-1 CHIC-Lys. Even though the wet adhesion strength of CHIC with 1 mgmL-1 and 10 mgmL-1 concentrations obviously increased, it was still lower than that of CHIC-Lys based on the same concentrations. In a typical case, the wet adhesion strength of 10 mgmL-1 CHIC-Lys could achieve to ~37 KPa which was a relative high value comparable to reported cases.31-32 The possible mechanism responsible for such considerable wet adhesion can be explained as blow.

Both CHIC and CHIC-Lys contain catechol groups so as to strong adhesion against

substrate based on the non-covalent interactions.26 When lysine molecules were introduced into the CHIC structure, the amino groups on lysine would form intramolecular hydrogen bonds with other polar groups such as phenolic hydroxyl, carbonyl and hydroxyl, resulting in improved wet adhesion because of arising viscoelasticity and cohesive energy. Furthermore, the rheology of the as-synthesized CHIC-Lys biomacromolecule was investigated. Firstly, the storage modulus (G’) and loss modulus G’’ for CHIC-Lys with


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different concentrations were measured with the change of shear stress at the room temperature. As shown in Figure 6A and Figure 6B, the G’ and G’’ values of the CHIC-Lys with 1 mgmL-1 and 10 mgmL-1 gradually decreased with increasing the shear stress, while 0.1 mgmL-1 CHIC-Lys exhibited low G’ and G’’ without obvious change. As shown in Figure S7, the viscosity of 0.1 mg·mL-1 CHIC-Lys was almost close to pure water, for which is a typical Newton liquid without obvious change in G'and G'. At the same time, it can be clearly seen that both G’ and G’’ gradually decreased with reducing the concentrations of the CHIC-Lys. Very interestedly, 10 mgmL-1 CHIC-Lys, we observed that G’’ value can become higher than its G’ value when shear stress is above the critical shear stress, as shown at the intersection point on the curves (Figure 6C), indicating a typical gel-sol transition state. However, for 1 mgmL-1 and 0.1 mgmL-1 CHIC-Lys, the G’ values were always lower than that of its G” values, indicating a typical sol state under such concentrations (Figure S2 and Figure S3). By contrast, for control 10 mgmL-1 CHIC sample, the G’’ value of it was always greater than G’ value (Figure 6C), indicating a typical sol feature without gel-sol transition feature. Besides, the G’ and G’’ values of CHIC-Lys were obviously larger than that of CHIC, indicating that CHIC-Lys has better mechanical strength than that of CHIC. Based on this, 10 mgmL-1 CHIC-Lys showed reversible gel-sol transition when shear stress cycled between low stress (0.2 Pa) and high stress (2 Pa) at the room temperature (Figure 6D). So, it can be concluded that CHIC-Lys is in a gel state with solid-liked behavior under static state or low stress shearing, while turns to a sol state with liquid-liked flow behavior under high stress shearing. Furthermore, typical shearing -thinning feature was clearly observed of CHIC-Lys with different concentrations under wide range of shear rates (Figure 6E). The shear-thinning



feature is commonly necessary as injectable auxiliaries when they are decorated onto surface of medical devices /instruments or singly used as fluid-liked medicine.33 Especially, the decrease of the viscosity at high stress shearing enables to weaken the friction between the auxiliaries and the tissues, which can effectively reduce the pains of patients. So, it is necessary to investigate the lubrication property of the CHIC-Lys. As shown in Figure 6F, the coefficient of friction (COF) of 1 mgmL-1 CHIC-Lys could be as low as 0.007, while it can achieve as high as ~0.25 for control CHIC sample with the same concentration.


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Figure 6 │ Rheological and friction tests for the as-synthesized biomacromolecules. (A) Storage modulus (G’), and (B) loss modulus (G’’) Vs shear stress (σ) for CHIC-Lys with different concentrations. (C) The evolution of G’ and G’’ of 10 mgmL-1 CHIC-Lys and CHIC as a function of the applied shear stress (σ= 0.2–2 Pa). (D) The evolution of G’ and G’’ as functions of time when cycled the shear stress between 0.2 Pa and 2 Pa. (E) Shear rate-dependent viscosity change for CHIC-Lys with different concentrations. (F) The change of coefficient of friction (COF) with time for PDMS slider against Ti6Al4V alloy lubricated by 1 mgmL-1 CHIC-Lys and CHIC under 1 N load at 1 Hz frequency at room temperature.

Figure 7 │ Haemostatic effect evaluation. (A) The photograph of Wistar rat as in vitro test model. The bleeding wound site with size of 20.2 cm2 on belly was covered by gauze with or without CHIC-Lys coating. (B) The photograph of the stripped gauze without CHIC-Lys coating from bleeding wound site after 5 minutes. (C) The photograph of the stripped gauze



with CHIC-Lys coating from bleeding wound site after 30 seconds. (D) The enlarged surface scanning electron microscope (SEM) image showing the transition area of CHIC-Lys coated needle. EDS mapping of Fe element (E) and C element (F) for same area in image (D). (G) The cross-sectional SEM image showing the CHIC-Lys coated needle. (H), (I) Photographs showing evaluation of haemostatic effect for bare needle. (J), (K) Photographs showing evaluation of haemostatic effect for CHIC-Lys-coated needle. Finally, we attempted to apply the as-synthesized CHIC-Lys biomacromolecule in biomedical field. Firstly, we investigated the hemostasis effect of the biomacromolecule when it was coated onto the gauze to treat the blooding mouse belly (Figure 7A). As shown in Figure7A, 7B, it is obvious find that the amount of bleeding on the CHIC-Lys-coated gauze was much lower than the blank gauze. As shown in Figure S4, the average blood loss on the blank gauze was ~0.78 g while the average blood loss on the CHIC-Lys modified gauze was only 0.18 g, implying the considerable application potential of CHIC-Lys as dressing. Subsequently, we attempted to coat the CHIC-Lys onto the surface of syringe needles to prepare functionally hemostatic needles. In a typical case, we coated the CHIC-Lys gel onto the surface of 5 mL syringe needles to prepare the modified needles. As shown in Figure 7G, the surface of the needle was uniformly wrapped by one layer of CHIC-Lys film after the solvent evaporating completely. Corresponding EDS mapping of Fe element (Figure 7E) and C element (Figure 7F) further proved this. As shown in Figure 7G, the average thickness of the coated CHIC-Lys film is ~100 μm. Besides, as illustrated in Figure 7(I) (K), compared to the simple needle, the CHIC-Lys-coated needle had a very good haemostatic effect. By using the simple needle, blood quickly oozing out at the injection site. The average blood loss by


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using simple needle was 135 L. Nevertheless, there was 100% haemostatic after incision of the femoral artery of rat by using CHIC-Lys-coated needle. The responsible mechanism for considerable hemostatic effect of CHIC-Lys can be well explained as follow. It was hypothesized that at the wound site the adhesive polymer could be strongly adhered onto the surrounding tissue and rapidly solidified to serve a bleeding-arrest barrier. Meanwhile, chitosan has a good role in promoting red blood cell aggregation, adhesion and aggregation of platelets, so that it has a good blood coagulation effect and can realize good hemostasis. Catechol can provide a strong adhesive at the wound site. And Lysine has good antibacterial action along with fast hydration. As a result, when the dry CHIC-Lys film encounters blood, it can rapidly hydrate but effectively adhere to the bleeding site, thereby achieving the effect of stopping bleeding. Subsequently, the swelling CHIC-Lys gel can stay in the wound site after pulling out the needles.

Figure 8 │ (A) Cell viability as a function of the concentration of CHIC-Lys. (B) The cell viability as a function of the incubation time of 0.1 mgmL-1 CHIC-Lys. After demonstrating the excellent hemostasis effect of the as-synthesized CHIC-Lys biomacromolecule while considering its potential application in biomedical filed, it is



extremely necessary to investigate its biocompatibility. In a typical case, we measured cell viability using CCK-8 assay. We incubated L-929 cells with different concentrations of CHIC-Lys and blank DMEM for 24 h. A wide range of concentrations of CHIC-Lys all had a positive effect on cell growth, as shown in Figure 8A. Meanwhile, 1 mgmL-1 CHIC-Lys significantly promoted cell proliferation. Especially, it was found that CHIC-Lys had no significant inhibitory effect on cell viability by prolonging the incubation time from 24 hours to 96 hours, as shown in Figure 8B. After 96 h incubation, the cell viability of 0.1 mgmL-1 CHIC-Lys was still more than 90%. Base on such results above, we can conclude that the as-synthesized CHIC-Lys has good biocompatibility.

4. Conclusions In this study, functional chitosan-catechol-lysine (CHIC-Lys) biomacromolecular wet adhesive was successfully synthesized. Compared with chitosan-catechol (CHIC) sample, the CHIC-Lys exhibits good swelling, wet adhesion, concentration-dependent gel-sol transition and obvious shear-thinning feature under wide concentrations range, along with good biocompatibility by the cell viability assay. Based on such considerable characteristics, CHIC-Lys can be successfully coated on the surface of syringe needle and gauze, and the decorated samples both show considerable haemostatic effect in test of rats venous bleeding. Overall, CHIC-Lys shows improved wet adhesion along with stress-sensitive rheological feature comparable to traditional chitosan (CS) and previous reported CHIC, making it as one kind of potential wet adhesive to be used in tissue engineering.


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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xxx. Detailed characterization by GPC for as-synthesized biomacromolecule; investigation of

G’ and G’’ change behavior for 1 mg·mL-1 CHIC-Lys and CHIC as a function of the applied shear stress; investigation of G’ and G’’ change behavior for 0.1 mg·mL-1 CHIC-Lys and CHIC as a function of the applied shear stress; quantitative study of the difference in blood loss between blank gauze and CHIC-Lys coated gauze.

Author Information Corresponding Author *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID Shuanhong Ma: 0000-0002-2144-4378 Feng Zhou: 0000-0001-7136-9233 Notes The authors declare no competing ﬁnancial interest.

Acknowledgements We gratefully acknowledge support from the National Key Research and Development Program of China (2016YFC1100401), the National Natural Science Foundation of China (51705507, 51605470) and the Bureau of International Cooperation, Chinese Academy of Sciences (121B62KYSB2017009).

Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))




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The table of contents entry: A new functional biomacromolecular wet adhesive as Chitosan-Catechol-Lysine (CHIC-Lys) is successfully synthesized by chemically grafting catechol moieties and L-lysine on chitosan backbones. The as-prepared CHIC-Lys shows improved wet adhesion strength and concentration-dependent gel-sol transition behavior. The CHIC-Lys coated needle and gauze both show considerable haemostatic effect in test of rats venous bleeding, exhibiting considerable application potential in biomedical field. Luyao Gao, Shuanhong Ma*, Jiajun Luo, Guangjie Bao, Yang Wu, Feng Zhou*, Yongmin Liang*

Synthesizing Functional Biomacromolecular Wet Adhesive with Typical Gel-Sol Transition and Shear-Thinning Features

TOC


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