Peptide-Functionalized Amino Acid Derived Pseudoprotein Based

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Peptide-Functionalized Amino Acid Derived Pseudoprotein Based Hydrogel with Hemorrhage Control and Antibacterial Activity for Wound Healing Jie Zhu, Hua Han, Faxue Li, Xueli Wang, Jianyong Yu, Xiaohong Qin, and Dequn Wu Chem. Mater., Just Accepted Manuscript • Publication Date (Web): 22 May 2019 Downloaded from http://pubs.acs.org on May 30, 2019

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Chemistry of Materials

Peptide-Functionalized Amino Acid Derived Pseudoprotein Based Hydrogel with Hemorrhage Control and Antibacterial Activity for Wound Healing Jie Zhu†, Hua Han†, Faxue Li*†,‡ , Xueli Wang,‡, Jianyong Yu‡, Xiaohong Qin*,† and Dequn Wu*†,‡

† Key Laboratory of Textile Science and Technology, Ministry of Education, College of Textiles, Donghua University, Songjiang District, Shanghai, 201620, China

‡ Innovation Center for Textile Science and Technology, Donghua University, Shanghai 201620, China.

ABSTRACT Developing permanent antibacterial and rapid hemostatic wound dressings with excellent biocompatibility is urgently needed and has always gains great attention. Here, a series of amino acid derived pseudoprotein consisting of poly(ester amide) (PEA) based hydrogel dressings and three types of cationic short peptides (RGDK, RRRFK and RRRFRGDK) are prepared. Compared with the antibacterial segments containing hydrogel scaffolds, the method of peptides modification of surface possesses the minimal usage of antibacterial moiety due to the effective 1

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contacting wound spots. Direct peptide RRRFRGDK (P3) conjugation to the hydrogel surface through an amidization reaction can enhance the antibacterial and hemostatic abilities with no or minimal outer appearance and inner morphology damage to the original hydrogels. The P3 functionalized hydrogel (Gel-g-P3) present excellent water uptake capacity, robust mechanical strength, enzymatic biodegradation, good hemocompatibility and cytocompatibility. Moreover, the Gel-g-P3 hydrogel has better adhesion capacities of blood cell and platelet, and exhibits shorter hemostasis time in mouse-liver injury model. Finally, the wound healing performance is evaluated in vivo using an infected wound model. The results show that Gel-g-P3 hydrogel has accelerated wound healing process, implying that the peptide functionalized PEA based hydrogels can be used as hemostasis agents and wound dressings for infected wounds.

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1.

INTRODUCTION Skin, as the largest organ and the first barrier in human body, plays vital roles in protecting the

body from external damage and microbial invasion.1, 2 However, the skin is easily damaged, and it needs a long time to repair once it suffers serious defects. During the repairing process, microorganisms would invade easily and form bacterial colonies on the wound site, leading to severe wound infection which would prolong healing process and even cause death due to the tissue damages.3-6 Besides, rapid hemorrhage control has also been one of the most urgent demands.7, 8 It is known that uncontrolled hemorrhage leads to more than 30% trauma deaths world-wide and more than half of these occur before emergency care can be reached.9, 10 Thus, employing wound dressings, as the hemostatic agent and bacterial barrier, to control the hemorrhage and infection rapidly and effectively is important for wound treatment. Modern wound dressings should not only meet the above demands but also should maintain a moist wound environment, absorb excess exudates, be removed easily, allow oxygen to permeate, relieve pain for patients and etc.11 Considering the aforementioned issues, hydrogels with inherent interconnected porous structure present remarkable potential as wound dressings.12,

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The characteristic porous

structure would allow the water/blood to flow into the hydrogels rapidly, thus endowing the materials with instantaneous exudate or blood absorption capacity.10, 14-16 Therefore, hydrogels are quite a physical barrier to blood loss which could promote the hemostatic plug formation. In order to avoid wound infection, hydrogels should possess good antibacterial property.2,

17, 18

Researchers have developed antibacterial dressings by incorporating antibacterial agents into 3



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hydrogels or directly using materials with inherent antibacterial activity. Compared with antibacterial agent loaded dressings, the latter possess persistent antibacterial activity. For instance, Wang et al. developed a biomimetic dopamine-modified ε-poly-L-lysine-polyethylene glycol-based hydrogel (PPD hydrogel) wound dressing for hemostatic and antibacterial application.19 This type of dressing took the advantage of forming a network barrier to realize the hemorrhage control and killed bacteria by the free amino groups. However, the authors found this antibacterial property exhibited degree of substitution (DS)-dependent behavior which could be impaired by a higher DS when synthesizing the PPD polymer. Cationic peptides provide a feasible way against bacterial infection by preferentially attracting to the negatively charged surfaces of bacteria over neutral zwitterionic mammalian cell membranes which then would result in the disruption of bacterial membranes, leaking the cytoplasmic

contents

and

ultimately

causing

bacteria

death.20-22

Furthermore,

the

positive-charged characteristics would enhance the interaction with blood cells and platelets through electrostatic interaction to induce blood cell aggregation, platelet activation, and coagulation.23 Particularly, studies have proved a RGD-containing peptide can provide biological ligands that can enhance cell adhesion and proliferation.24,

25

Although cationic peptides have

been reported to possess excellent antibacterial activities, possible side effects such as hemolysis and cytotoxicity may hamper their clinical application.26 To address these issues, peptide-conjugated polymers and scaffolds have been utilized without changing the antibacterial properties.24 Sun et al. listed a series of studies on antimicrobial peptides-polymer conjugates with excellent a broad spectrum of antibacterial property, high 4


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selectivity and low cytotoxicity.20 Compared with peptide-conjugated polymers, modification of a surface using peptides can enhance functionality of scaffold surface more efficiently, such as cell-surface interaction. Besides, direction peptide modification would have no or minimal damage on the outer appearance and the inner morphology.27 Deng et al. prepared vitronectin peptide-presenting hydrogel for application in cell therapy and bone tissue engineering.24 Inspired by those designs, antibacterial peptide conjugated hydrogels could be considered as hemostasis agents and wound dressings. Degradable hydrogel wound dressings are attracting increasing interest because this type of hydrogels usually consists of highly biocompatible macromolecules.28 Recently, we have successfully synthesized the amino acid derived random poly(ester amide)s (PEAs) which are proved to be biocompatible and biodegradable due to the enzyme recognizable segments on their backbones (e.g. the ester and amide blocks).29 Importantly, the degradative small molecules of PEA polymers including amino acids based molecules are highly biocompatible. Here, we designed peptide conjugated hybrid hydrogel dressings with hemorrhage control and antibacterial properties. In detail, the hybrid hydrogels were formed by methyl methacrylate lysine (Lys) based PEA (4-Lys-4-MA), methyl ether methacrylate methoxy polyethylene glycol (mPEG-MA) and 2-aminoethyl methacrylate hydrochloride (AEMA) via photo crosslinking method, and the peptide was then conjugated to the hydrogel surface through an amidization reaction. The morphologies of freeze-dried hydrogels were observed by scanning electron microscopy (SEM). Swelling, mechanical properties, biodegradation, hemocompatibility, cytocompatibility and antibacterial property of hydrogels were characterized. Finally in vivo 5



bleeding control and wound healing were evaluated and results proved that the hydrogels could achieve rapid hemorrhage control and efficient infection prevention.

2. RESULTS AND DISCUSSION Synthesis of Monomer and Hydrogels. A series of precursor 4-Lys-4 (x-Lys-y, where x and y represent the methylene in the diacid monomer and diol monomer, respectively) based hydrogel dressings with hemorrhage control and antibacterial properties were prepared in this study. Firstly, 4-Lys-4 was synthesized with molecular weight of 1800 g/mol (polydispersity of 1.37). The methyl methacrylate 4-Lys-4 (4-Lys-4-MA) and mPEG-MA macromers were synthesized via nucleophilic substitution of 4-Lys-4 and methacrylic anhydride (MA), mPEG and methacryloyl chloride (MAC), respectively (Scheme 1a and b). To confirm the structure of the synthesized 4-Lys-4 and 4-Lys-4-MA, 1H NMR spectra were characterized as shown in Figure 1a. The characteristic peaks at chemical shift at 7.56 and 7.20 ppm from Lys-4 monomer were attributed to H in the benzene ring which disappeared in the spectra of 4-Lys-4 and 4-Lys-4-MA polymers.30 Besides, it was noted that new peaks at 5.56 and 5.33 ppm appeared in the spectra of 4-Lys-4-MA which were attributed to the -CH=CH2. The DS of the resulting 4-Lys-4-MA was calculated as 32.5%. mPEG-MA was synthesized according to the method in the previous study,12 and the 1H NMR spectra were shown in Figure S2 (Supporting Information). Secondly, to improve the hemorrhage control and antibacterial abilities, three types of peptides with the sequences of RGDK (designated as P1), RRRFK (P2) and RRRFRGDK (P3) were synthesized (purity: >95%). The HPLC and FT-IR results of the peptides were shown in 6


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Figure S3-6 and Table S1-3 (Supporting Information). Finally, the hydrogels were formed by photo-crosslinking using ammonium persulfate (APS) as illustrated in Scheme 1c. The feed ratio of 4-Lys-4-MA, mPEG-MA and 2-aminoethyl methacrylate hydrochloride (AEMA) precursors was listed in Table 1. In this hydrogel system, 4-Lys-4-MA served as the major network of hydrogels and mPEG-MA could endow the hydrogels with a more hydrophilic characteristic, while the introduction of AEMA could provide more free amino groups for the conjugation of peptides. As there were unreacted and free amino groups of the hydrogel surface, P3 could be further conjugated to the surface of the hybrid hydrogel Gel-2 fabricating Gel-g-P3 with the conjugation density of 2.3 ± 0.3 pmol/mm2. The FT-IR spectra of the resultant hydrogels were shown in Figure 1b. It could be seen that the peaks at about 1745 cm-1, 1650 and 1540 cm-1 could be observed in all hydrogels and the peaks at the range 700-800 cm-1 could be found in the hydrogel Gel-g-P3 which were attributed to the benzene ring bending vibration of the P3, confirming the successful conjugation of P3 to the hydrogel surface.

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Scheme 1. Schematic representation for the preparation of 4-Lys-4 based hydrogels. (a) Synthesis of 4-Lys-4-MA polymer via nucleophilic substitution using 4-Lys-4 and MA. (b) Synthesis of mPEG-MA polymer. (c) Preparation of hybrid hydrogels. 4-Lys-4-MA, mPEG-MA and AEMA precursors with different feed ratios were mixed and the hybrid hydrogels were then formed by photo-crosslinking using APS. After that, P3 was grafted onto the hydrogel surface in the presence of NHS and EDC. In this hydrogel system, 4-Lys-4-MA served as the major network of hydrogels and mPEG-MA could endow the hydrogels with a more hydrophilic 8


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characteristic, while the introduction of AEMA could provide more free amino groups for the conjugation of peptides.

Figure 1. Preparation of hydrogel precursors, peptides and hydrogels. (a) 1H NMR spectra of Lys-4 monomer, 4-Lys-4 and 4-Lys-4-MA polymers. New peaks at 5.56 and 5.33 ppm (Peak m) appeared in the spectra of 4-Lys-4-MA which were attributed to the -CH=CH2. (b) FT-IR spectra of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels. Inset showed 9



the magnified FT-IR spectra with the wavenumbers from 900-700 cm-1. (c) Morphologies of the freeze-dried hydrogels characterized by SEM. i: 4-Lys-4-MA, ii: 4-Lys-4-MA/AEMA, iii: Gel-1, iv: Gel-2, v: Gel-3 and vi: Gel-g-P3. The morphological structure of hydrogels could be controlled by varying the feed ratio of components. The pore size of the Gel-1, Gel-2 and Gel-3 hydrogels was gradually decreased. After the conjugation of P3, the pore size of the Gel-g-P3 hydrogel was a bit larger than that of no grafted one.

Morphology and Swelling Ratio of Hydrogels. The morphology of the freeze-dried hydrogels was studied by SEM (Figure 1c). Images showed all the hydrogels had interconnected porous structure and the pore size of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2 and Gel-3 hydrogels was about 6.15 ± 1.53, 4.22 ± 1.73, 10.73 ± 3.54, 6.78 ± 1.32 and 4.71 ± 1.10 μm, respectively (Figure S7, Supporting Information). It was reported the porous structure can be explained by the elimination of water from the hydrogels.31 As the 4-Lys-4-MA polymer was hydrophobic which could decrease the interaction with water molecules, the 4-Lys-4-MA and 4-Lys-4-MA/AEMA hydrogels keep to remain the original structure, resulting into the denser structure (as circled in red).32 In contrast, the hybrid hydrogels containing mPEG-MA components displayed more regular porous structure with continuous walls. The results indicated the morphological structure of hydrogels could be controlled by varying the feed ratio of components. The pore size of the Gel-1, Gel-2 and Gel-3 hydrogels was gradually decreased caused by the higher cross-linking density (Table S4, Supporting Information). Besides, after the conjugation of P3 on the hydrogel surface, the pore size of the Gel-g-P3 hydrogel was a bit larger 10


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(8.01 ± 2.03 μm) and less uniform than that of Gel-2 hydrogel. This was because the hydrogen bonding interaction between P3 and water molecules had been enhanced, which led the Gel-g-P3 hydrogel to absorb a larger amount of water and accelerated diffusion of water into hydrogel matrix.31 Benefited from the interconnected and porous structure, the hydrogels could display water uptake capacity which might be utilized as an absorbing agent of wound exudate for reducing the bacterial infection and promoting wound healing.33 On the other hand, the water absorption property could enhance the hemorrhage control ability by concentrating the clotting factors.34 The swelling ratio, as a quantitative index of water uptake capacity, was investigated at predetermined time (Figure 2a). The 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2 and Gel-3 hydrogels had an average swelling ratio of 190.41%, 531.34%, 1537.22%, 1210.62% and 930.34%, respectively after 10 min. It could be seen that the introduction of AEMA and mPEG-MA components could significantly improve the water absorbing ability of hydrogels compared with the pure 4-Lys-4-MA hydrogel. In addition, the hybrid hydrogels (Gel-1, Gel-2 and Gel-3) with more mPEG-MA contents exhibited larger swelling ratio. This phenomenon could be explained by the inner structure of hydrogels; hydrogels with more 4-Lys-4-MA contents exhibited more compact porous structure which would decrease the water uptake capacity.35 Moreover, the swelling ratio of the Gel-g-P3 hydrogel was 1462.62% at 10 min which was a little higher than that of Gel-2 hydrogel, illustrating the conjugation of P3 could increase the water absorbing property.36

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Figure 2. Water uptake, biodegradation and mechanical properties of hydrogels. (a) Swelling ratio of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels in PBS buffer (pH 7.4, 0.1 M). (b) Enzymatic biodegradation of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels versus time. Enzyme: trypsin at a concentration of 0.15 mg/mL in PBS buffer (pH 7.4, 0.1 M). (c) Morphologies of the freeze-dried 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels after 3 days of biodegradation. Scale bars are 10 μm. (d) Compressive modulus of hydrogels. i: 4-Lys-4-MA, ii: 4-Lys-4-MA/AEMA, iii: Gel-1; iv: Gel-2, v: Gel-3, vi: Gel-g-P3. Rheological performance of (e) 4-Lys-4-MA, (f) 4-Lys-4-MA/AEMA hydrogels, (g) Gel-1, (h) Gel-2 and Gel-g-P3 and (i) Gel-3 hydrogels.

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Results are presented as mean ± standard deviation and * indicates significant difference (p < 0.05); ** indicates significant difference compared with all other conditions (p < 0.01).

Biodegradation and Mechanical Properties of Hydrogels. The trypsin catalyzed biodegradation property of the hydrogels was examined in terms of their weight loss and SEM morphology (Figure 2b and c). The weight loss of the 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2 and Gel-3 hydrogels were about 49.16%, 32.02%, 28.92%, 23.33% and 16.11%, respectively, over a period of 168 h (Figure 2b), while the weight of the corresponding hydrogels in the PBS buffer without enzyme had little change after 168 h (Figure S8, Supporting Information). Besides, the Gel-g-P3 had average weight loss of 25.10% at 168 h which was a little higher than that of the Gel-2 hydrogel because P3 on the surface of hydrogel was easier to expose to the enzyme solution and underwent degradation. The morphology of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels after biodegradation was also different with that before degradation; apparently, a part of the porous structure was gradually broken (Figure 2c). It is worth noting that the biodegradation rate of the 4-Lys-4-MA hydrogel is much faster than that of the other hydrogels and the biodegradation rate of the Gel-1, Gel-2 and Gel-3 hydrogels would be lower with the increasing of the mPEG-MA amounts. As trypsin could catalyze the breakdown of the amide bond, the hydrogels with a higher proportion of 4-Lys-4-MA would have a higher degradation rate.37 The hydrogels should also demonstrate enough mechanical strength to not hinder their practical application for hemostasis and wound healing. The compression properties of the 13



4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels were investigated (Figure 2d). The 4-Lys-4-MA hydrogel had the highest compressive modulus and followed by the 4-Lys-4-MA/AEMA hydrogel. The compressive modulus of the Gel-1, Gel-2 and Gel-3 hydrogels was increased with the increasing of 4-Lys-4-MA amounts due to the more compact structure which was consistent with the SEM observation. The rheological properties of the hydrogels in Figure 2e-i were also consistent with the compression results. The 4-Lys-4-MA and 4-Lys-4-MA/AEMA hydrogels had the highest storage modulus (higher than 100 Pa), and the storage modulus of the other hydrogels varied at the order of Gel-3 > Gel-2 > Gel-1. All the above results indicated that the hydrogels exhibited good mechanical properties to protect wounds in the practical application as wound dressings. Hemocompatibility and Cytocompatibility of the Peptides and Hydrogels. Ideal biomaterials should be nontoxic or minimally toxic to the human body, and induce no or little hemolysis. For this reason, the blood compatibility and cytocompatibility of the peptides (P1, P2 and P3) were investigated (Figure 3). Hemolysis assay is the most common method to characterize the hemolytic activity. Insets showed the obtained supernatants of the peptide groups, negative Tris-HCl and positive Triton X-100 groups (Figure 3a-c). It was found that the Tris-HCl presented light yellow color, and the peptide groups displayed the color of peptide solutions which were gradually darkened but not red with the increasing of peptide concentration, demonstrating peptides would induce negligible hemolysis. In contrast, the Triton X-100 group showed a bright red color. As shown in Figure 3a-c, the P1, P2 and P3 exhibited the hemolysis ratio of 0.29%, 0.31% and 0.30%, respectively at a concentration of 1.56 μg/mL (P1: 3.3 μM; P2: 14


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2.1 μM; P3: 1.4 μM), and that values would increase to 1.28%, 1.78% and 1.49%, respectively, at the highest concentration of 200 μg/mL (P1: 421.9 μM; P2: 262.8 μM; P3: 183.5 μM). As seen in Figure S9 (Supporting Information), compared with the short peptides in other literatures, the prepared peptides (P1, P2 and P3) in this study had very low hemolytic activities. In general, the hemolytic activity can be affected by the peptide length and the hydrophobicity (hydrophobic residues).38 As the prepared peptides are short peptides, the peptide length will generate little difference in hemolysis activity. Considering the hydrophobic interaction, the P2 and P3 with hydrophobic residues might slight penetrate into the interior neutral erythrocyte cell membranes due to hydrophobic interaction, resulting into a higher hemolysis value compared with P1, and P2 had a slightly higher hemolysis effect than P3.39, 40 Overall, the results revealed the peptides induced little hemolysis even at a concentration of 200 μg/mL which would exhibit insignificant hemolytic activity for in vivo application. The cytotoxicity of P1, P2 and P3 peptides was also tested by MTS assay after 24 and 48 h (Figure 3d-f). The cell viability of peptides was related to the concentration of peptide solution; when bearing a concentration of 50 μg/mL, the peptides had the lowest cytotoxicity, and the cell viability would have a slight decrease with the increasing of concentration. In detail, even at a concentration of 200 μg/mL, the cell viability of P1, P2 and P3 peptides after 24 and 48 h were 83.97% and 81.79%, 79.70% and 77.85%, 86.88% and 82.72%, respectively. It is obvious that all peptide groups exhibited low cytotoxicity by quantitative analysis. Besides, the live/dead staining micrographs exhibited there were a great number of living cells (green) and nearly no apoptotic cells (red) in the groups at a concentration of 50 μg/mL compared with 200 μg/mL 15



after 24 h (Figure 3g). Overall, the results demonstrated the peptides are biocompatible which allowed them in vivo application. As the hydrogels will contact with the blood and human body directly when they are applied as wound dressings, their hemolytic activity and cytocompatibility were characterized (Figure 4a and b). The hydrogel groups presented light yellow (Figure 4a) and the hemolysis ratio of the 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels’ powder suspensions was about 0.70%, 1.03%, 1.79%, 1.59%, 1.40% and 1.68%, respectively at a concentration of 500 μg/mL (Figure 4b). The hemolysis ratio could be further increased to 2.55%, 2.70%, 3.82%, 3.34%, 3.03% and 3.53%, respectively at a concentration of 4000 μg/mL. As hemolysis is the destruction of erythrocyte cells in the blood and maximum 5% hemolysis ratio is accepted for hemocompatible materials, the hemolysis results revealed that the hydrogels were excellent candidates for hemostasis agents and wound dressings. The MTS assay results and representative micrographs showed that all the hydrogels had nearly no cytotoxicity (higher than 85%) on L929 Fibroblast cells after 24 h (Figure 4c and d). In addition, a live/dead cell viability assay was also conducted to investigate the influence of hydrogels on L929 Fibroblast cells (Figure 4e) and it could be observed almost all the cells were stained green which confirmed the excellent biocompatibility of the hydrogels.

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Figure 3. The blood compatibility and cytocompatibility of peptides. Hemolytic activity of (a) P1, (b) P2 and (c) P3 peptides as a function of concentration. The peptides induced little hemolysis even at a concentration of 200 μg/mL which would exhibit insignificant hemolytic activity for in vivo application. Cell viability of (d) P1, (e) P2 and (f) P3 peptides as a function of concentration for L929 fibroblast cells. The cell viability would have a slight decrease with the increasing of concentration. (g) Live/dead staining of L929 fibroblast cells after culturing with P1, P2 and P3 peptides with concentration of 50 μg/mL (i-iii) and 200 μg/mL (iv-vi), and TCP was used as control. There were a great number of living cells (green) and nearly no apoptotic 17



cells (red) in the groups at a concentration of 50 μg/mL compared with 200 μg/mL after 24 h. Scale bars are 200 μm.

Figure 4. The blood compatibility and cytocompatibility of the hydrogels. (a) and (b) Hemolytic activity of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-1, Gel-2, Gel-3 and Gel-g-P3 hydrogels as a function of hydrogel powder concentration. (c) Cell viability of hydrogels on L929 fibroblast cells. A: 4-Lys-4-MA, B: 4-Lys-4-MA/AEMA, C: Gel-1, D: Gel-2, E: Gel-3, F: Gel-g-P3. (d) Representative micrographs of L929 Fibroblast cells incubated with hydrogels after 24 h. i: 4-Lys-4-MA, ii: 4-Lys-4-MA/AEMA, iii: Gel-1, iv: Gel-2, v: Gel-3, vi: Gel-g-P3. Scale bars are 100 μm. (e) Live/dead staining of L929 fibroblast cells after culturing with hydrogels. i: 4-Lys-4-MA, ii: 4-Lys-4-MA/AEMA, iii: Gel-1, iv: Gel-2, v: Gel-3, vi: Gel-g-P3. Scale bars are 200 μm. 18


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Antibacterial evaluation of Peptides and Hydrogels. The antibacterial properties of the peptides against the gram-negative bacteria Escherichia coli (E. coli) and the gram-positive bacteria Staphylococcus aureus (S. aureus) were investigated using the optical density (OD) value at 600 nm (Figure 5a-f) as a function of peptide concentration based on the minimum inhibitory concentrations (MICs, Table S5, Supporting Information). Compared with the untreated bacterial suspensions (Figure S10 and S11, Supporting Information), the values at OD 600 nm for P1 rose more slowly against both E. coli and S. aureus, especially for that with a higher peptide concentration. Interestingly, the values at OD 600 nm for P2 and P3 groups were kept decreasing after 3 h and 2 h, respectively even at a concentration of 50 μg/mL, indicating that P2 and P3 peptides possessed a better bacterial inhibition capacity compared with P1. What’s more important, P3 had a faster therapy effect for inhibiting bacteria growth at a lower concentration. To further compare the antibacterial property of P1, P2 and P3, the bacterial growth inhibition capacity was studied by the colony counting method after being treated for 8 h (Figure 5g). Consistently with the OD results, P1 exhibited the lowest killing bacteria capacity among the three types of peptides. The log colony forming unit (CFU) reduction of E. coli was 0.47, 0.56, 0.72 and 0.89 at the concentration of 50, 100, 150 and 200 μg/mL, respectively, which corresponded to a promising efficacy of 66.3%-87.2%; the log CFU reduction of S. aureus was 0.61 0.71, 0.80 and 1.01 at the concentration of 50, 100, 150 and 200 μg/mL, respectively, which corresponded to a promising efficacy of 75.6%-90.3%. In great contrast, P3 achieved 1.67, 2.96, 19



3.18 and 3.25 log CFU reduction of E. coli, 2.51, 3.20, 3.81 and 4.20 log CFU reduction of S. aureus at the concentration of 50, 100, 150 and 200 μg/mL, respectively, which corresponded to a promising efficacy of 97.88%-99.99%. It was reported that cationic peptides with more than two net charges were prone to bind to the negative membrane of bacteria and other microorganisms.26 In addition, increasing the net charge within a limited range (net charges from +2 to +9) could enhance the antibacterial activity, along with a moderate increase of hemolysis.41 However, continuously increasing the cationic charge would result in rapid increase of hemolytic activity. Besides, the hydrophobic residue was another essential factor which played a critical role in creating a pore in the microbial membrane.42 After attaching onto microbial membrane via electrostatic attraction, the hydrophobic residues in peptides began to interact with hydrophobic segment of lipid bilayer, resulting in disruption of the microbial membrane. In this study, P2 and P3 with hydrophobic residues and net charges of +4 had a better antibacterial activity than P1 (net charges of +2) in physiological environment. The live/dead bacteria viability assay of E. coli and S. aureus before and after incubation with P3 at a concentration of 50, 100, 150 and 200 μg/mL for 8 h was carried out. (Figure 5h and i). The bacteria were stained with a propidiumiodide (PI) red dye and a SYBR Green (SG) dye. In each group, the left image showed the bacteria existed in the bacterial suspension (stained green) and the right one showed the dead bacteria (red). There were a large amount of bacteria (green) in all groups, however, almost all the bacteria fluoresced red in the P3 treated groups, revealing the excellent antibacterial activities of P3. To further elucidate the antibacterial mechanism of P3, the morphological changes of E. coli and S. aureus before and after incubation 20


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with P3 at a concentration of 200 μg mL-1 for 4 and 8 h were investigated (Figure 5j and k). The untreated E. coli and S. aureus had rod and round shapes with smooth surfaces, respectively, whereas the cell walls and membranes of the bacteria became rough and were efficiently disrupted as circled after 4 h. Generally, the positively charged characteristic of P3 is responsible for engaging E. coli and S. aureus bacteria.21, 43, 44 For P3, repeated arginine sequences with a high content of guanidine groups can bind to the outer leaflet membrane via strong hydrogen bonds and electrostatic attraction.45 Then, the hydrophobic benzene ring of the peptide would insert into the lipid portion of the membrane to break the bacteria cell walls and cause cell death.21, 46 It is well known that the wound healing process might be suffered from bacterial infection which will delay the wound healing.2,

17

To address this issue, antibacterial dressings are

designed to inhibit the bacteria growth. The antibacterial activity of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels was assessed qualitatively against E. coli and S. aureus (Figure 6a and b). No inhibition zone could be observed in the 4-Lys-4-MA group, and there was a small inhibition zone in 4-Lys-4-MA/AEMA and Gel-2 groups which might be caused by the free amino groups from AEMA component. In contrast, a clearer and larger inhibition zone was formed in Gel-g-P3 group towards E. coli and S. aureus (Figure 6a). Besides, it was noteworthy there were little bacteria adhered on hydrogels, especially for the Gel-2 and Gel-g-P3 hydrogels after the antibacterial test (Figure 6c). It was believed that the number of bacteria adhered was dependent on the hydrophilic character of surface; that was, less bacteria would adhere to the surface with the increase of the hydrophilic character for the hydrogels.47 As 21



a result, the hydrogel wound dressings could not be easily contaminated because the bacteria were not easy to adhere to the surface of hydrogels owing to the moist characteristic. The antibacterial effect of Gel-g-P3 hydrogel was illustrated as shown in Figure 6d. When applied at a bacteria-infected wound site, the positively charged grafted P3 could interact with the negatively charged bacterial cell membranes via electrostatic interaction to cause the bacteria death, and the dead bacteria subsequently could be repellent to the Gel-g-P3 hydrogel surface due to the moist character.47

22


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Figure 5. Antibacterial evaluation of peptides against E.coli and S. aureus. OD growth curves of bacterial suspensions treated with peptides at different concentrations (50, 100, 150, 200 μg/mL) versus time in PBS (pH 7.4, 0.1 M). E. coli suspensions treated with (a) P1, (b) P2 and (c) P3, S. aureus suspensions treated with (d) P1, (e) P2 and (f) P3. (g) The bacterial growth inhibition against E. coli and S. aureus investigated by the colony counting method. The bacterial suspensions treated with P1, P2 and P3 at different concentrations after 8 h were collected and 23



diluted with PBS to form bacterial colonies using an agar diffusion assay. Live/dead bacterial viability assay of (h) E.coli and (i) S. aureus in untreated group (control) and P3 groups after 8 h. i: 50 μg/mL, ii: 100 μg/mL, iii: 150 μg/mL, iv: 200 μg/mL. Scale bars are 50 μm. SEM images of (j) E. coli and (k) S. aureus before and after being treated with P3 at a concentration of 200 μg/mL after 4 and 8 h. The circled indicated deformed and damaged sites on the bacteria cell walls after treatment of P3.

Figure 6. Antibacterial assessment of hydrogels against E.coli and S. aureus. (a) Optical images of antibacterial activity of hydrogels against E. coli and S. aureus after 24 h. (b) The measured inhibition zone of hydrogels against E. coli and S. aureus after 24 h. (c) Morphologies of the freeze-dried 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels after antibacterial test. (d) The antibacterial illustration of Gel-g-P3 hydrogel. The positively charged grafted P3 can interact with the negatively charged bacterial cell membranes via electrostatic interaction to 24


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cause the bacteria death, and the dead bacteria subsequently can be repellent to the Gel-g-P3 hydrogel surface.

In vitro Blood Clotting Performance of Hydrogels. The blood clotting capacity of hydrogels was assessed to evaluate the hemostatic property of hydrogels (Figure 7). In this study, in vitro whole blood clotting of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels was investigated for measuring their blood clotting index (BCI). Gauze, which was widely used as a clinical hemostatic agent, was utilized as control. In this test, a higher BCI value indicated a slower clotting rate.12 As shown in Figure 7a, the BCI values of gauze group were 88.61% and 76.42% in 5 and 10 min, respectively. Interestingly, the 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels showed much lower BCI values than the gauze group. In addition, among the four types of hydrogels, the Gel-g-P3 hydrogel had the lowest BCI value which was 35.22% and 21.53% at 5 and 10 min, respectively; the 4-Lys-4-MA had the highest BCI value which was 52.15% and 38.94% at 5 and 10 min, respectively. It could be found the blood clotting capacity was related to the water absorption ability of hydrogels; a higher water absorption capacity would lead to a better blood clotting ability. Moreover, the result showed introducing P3 could further enhance the blood clotting ability. The images of gauze and hydrogels exhibited all samples absorbed bloods after 5 min (Figure 7b). In order to explore the hemostatic mechanism of the hydrogels, the adhesion of blood cells and platelets on the hydrogels was characterized, and gauze was also used as control (Figure 7c and d). There were many blood cells adhering to the hydrogel surfaces or the walls of pores for all the hydrogels 25



compared with the gauze group (Figure 7c). Furthermore, each hydrogel showed platelets adhesion, whereas very few platelets were adsorbed to gauze. With the introduction of P3, the Gel-g-P3 hydrogel showed a higher degree of platelet adhesion (Figure 7d). On one hand, the hydrogels with interconnected porous structure could first absorb a large amount of blood quickly, resulting in the platelet and blood cell absorption, which would induce the blood clotting. On the other hand, the positive-charged peptide could induce hemostasis through electrostatic attraction to adhere blood cells, platelets, and plasma fibronectin.48

Figure 7. In vitro blood clotting performance of hydrogels. (a) The BCI of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels and gauze within 5 and 10 min. (b) 26


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Photographs of samples used in vitro whole blood clotting test after 5 min. (c) SEM images of blood cells adhesion on the hydrogels and gauze. There were many blood cells adhering to the hydrogel surfaces or the walls of pores for all the hydrogels compared with the gauze group. Scale bars are 40 μm. (d) SEM images of platelet adhesion on the hydrogels and gauze. Scale bars are 25 μm. The hydrogels with interconnected porous structure could first absorb a large amount of blood quickly, resulting in the platelet and blood cell absorption, and the positive-charged peptide could induce hemostasis through electrostatic interaction to adhere blood cells, platelets, and plasma fibronectin.

In vivo Hemostatic Properties of Hydrogels. In vivo hemostatic properties of 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels were further evaluated using a mouse liver model, and gauze was also used as control (Figure 8). For the mouse liver model, the mouse liver was first exposed and cleaned. After inducing bleeding, the sample was placed to cover the bleeding site (Figure 8a). Seen from Figure 8b, a filtration paper which was changed every 15 s was employed for absorbing the blood and the results indicated hydrogels could stop bleeding in a shorter period which were more effective on hemostasis compared with gauze. Besides, the hemostatic time was counted, and 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels showed average hemostatic time of 78 s, 72 s, 65 s and 57 s, respectively, whereas gauze group had the longest hemostatic time more than 3 min (Figure 8c). The blood loss from the livers in 3 min was weighed as shown in Figure S12 (Supporting Information). The Gel-g-P3 had a favorable hemostasis effect compared with some commercial hemostatic materials listed in 27



Table S6 (Supporting Information). The above results demonstrated that the hydrogels had much better hemostatic capacity than the traditional gauze. Moreover, Gel-2 and Gel-g-P3 presented better hemostatic ability than 4-Lys-4-MA and 4-Lys-4-MA/AEMA hydrogels, and the conjugation of P3 on hydrogel surface could promote the hemostatic ability of hydrogel. In general, there are three strategies for inducing hemostasis, that is, (i) enhancing the interaction of proteins of the coagulation cascade; (ii) providing a matrix for clot formation; and (iii) improving adherence to tissues to seal bleeding wounds.49 As illustrated in Figure 8d, the hydrogel worked as a physical barrier and a matrix for concentrating the blood to entrap aggregated hemocytes, which fulfilled the last two of the above strategies. In detail, after inducing bleeding, the hydrogels with interconnected porous structure could inhibit blood loss and absorb the blood at rapid absorption speed.34 During this process, blood cells and platelets could be absorbed into hydrogels which could enhance the generation of thrombin, thus accelerated coagulation. As mentioned before, the grafted positive-charged P3 on the hydrogel surface would improve the interaction with blood cells and platelets through electrostatic attraction to induce blood cell aggregation and platelet activation, which fulfilled the first of the above strategies.10 Therefore, the conjugation of P3 in the Gel-g-P3 hydrogel system could be given rise to coagulation acceleration.

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Figure 8. In vivo hemostatic assessment of hydrogels. (a) The procedures for a liver model in a mouse model. The mouse liver was first exposed and induced bleeding, the sample was then placed on the bleeding site. (b) The evaluation of hemostatic ability of the gauze and 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogels using a filtration paper which was changed every 15 s. (c) Hemostatic time in the mouse liver model. (d) Scheme representation of the hemostatic process of Gel-g-P3 hydrogel. The hydrogels could act as a physical barrier to blood loss and absorb the blood at rapid absorption speed, and blood cells and platelets could be absorbed into hydrogels via electrostatic interaction.

In vivo Wound Healing Performance. The wound healing performance of the hydrogels was evaluated by using in vivo test. In this experiment, the full-thickness wound was created and S. 29



aureus bacterial suspensions were then injected on the injured site for 1 h. After that, the wound in each mouse was covered with gauze (control group), gauze soaked with P3 in PBS (100 μL, 200 μg/mL), 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogel samples, respectively. The wound tissues were collected to determine the number of bacteria after 3 days (Figure S12, Supporting Information). The bacteria colonies in the P3 and hydrogel groups were less than the gauze group, especially the Gel-g-P3 group, indicating its effective bacteria-eliminating function. The P3, 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 groups achieved 2.64, 0.79, 1.11, 1.23 and 2.95 log CFU reduction, respectively. Representative photographs of the healing process were shown in Figure 9a. After 7 days’ treatment, the wound treated with only gauze showed a dark, dried and wrinkled appearance and it was even worse in the following days. Comparatively, significant differences in wound appearance could be observed between the control group and other groups; there was no obvious dark scar in the P3 and hydrogel groups. The wound size was measured in 15 days and the wound area reduction was quantitatively illustrated in Figure 9b. After being treated for a week, the P3 soaked gauze and hydrogels showed higher wound area closure ratio (P3: 38.0%; 4-Lys-4-MA: 39.4%; 4-Lys-4-MA/AEMA: 43.6%; Gel-2: 50.4%; Gel-g-P3: 58.9%) than control group (22.4%). Moreover, the wounds in the Gel-2 and Gel-g-P3 groups nearly healed after 15 days (Gel-2: 88.5%; Gel-g-P3: 93.7%) which were significantly faster than other groups (control: 58.0%; P3: 79.2%; 4-Lys-4-MA: 81.5%; 4-Lys-4-MA/AEMA: 84.4%). The inflammatory response was investigated using the serum inflammation indexes including tumor necrosis factor-α (TNF-α), interleukin-1 IL-1β, interleukin-6 (IL-6) and monocyte 30


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chemoattractant protein-1 (MCP-1) by ELISA analysis after 15 days. As shown in Figure 9c-f, there was a significant decrease of all the values in Gel-2 and Gel-g-P3 groups compared with the control, P3, 4-Lys-4-MA and 4-Lys-4-MA/AEMA groups (p < 0.01). Moreover, Gel-g-P3 group showed a less severe inflammatory response than Gel-2 group which was consistent with the wound healing result, demonstrating its excellent antibacterial and accelerated wound healing effects on infected wounds. Hemotoxylin and eosin (H&E) staining was used for histomorphological examination of the wound healing after 15 days. The histological results indicated nearly no epidermis structure could be found in the control, P3, 4-Lys-4-MA and 4-Lys-4-MA/AEMA groups, and different levels of inflammatory cells could be observed in these groups (Figure 10). Comparatively, less inflammatory cells were seen and more newly produced collagen was deposited in Gel-2 and Gel-g-P3 groups after 15 days. As known, low level inflammation will accelerate wound healing process especially in the early wound healing stage. Therefore, Gel-g-P3 hydrogel could accelerate wound healing process benefited from the integration properties of moisture environment and antibacterial property, suggesting its huge potential as wound dressings.

31



Figure 9. In vivo wound healing evaluation by an infected and full-thickness wound model. (a) Representative photographs of the gauze (control), P3 soaked gauze, 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3 hydrogel treated wounds. (b) Evaluation of the wound area closure in 15 days. Expression level of TNF-α (c), IL-1β (d), IL-6 (e) and MCP-1 (f) after 15 days. Results are presented as mean ± standard deviation and * indicates significant difference (p < 0.05); ** indicates significant difference compared with all other conditions (p < 0.01).

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Figure 10. H&E staining of wound sections treated with (a) Gauze, (b) P3, (c) 4-Lys-4-MA, (d) 4-Lys-4-MA/AEMA, (e) Gel-2 and (f) Gel-g-P3 after 15 days. There was nearly no epidermis structure appeared in the control, P3, 4-Lys-4-MA and 4-Lys-4-MA/AEMA groups, and different levels of inflammatory cells could be observed. Comparatively, less inflammatory cells were seen and more newly produced collagen was deposited in Gel-2 and Gel-g-P3 groups. The black scale bars are 200 μm; the blue scale bars are 100 μm.

3. CONCLUSION In this study, a series of hydrogels based on PEA, mPEG and AEMA were prepared. To improve the hemorrhage control and antibacterial abilities, peptide with the sequence of RRRFRGDK (P3) was conjugated to the hydrogel surface. In this hydrogel system, 4-Lys-4-MA 33



served as the major network of hydrogels and mPEG-MA could endow the hydrogels with a more hydrophilic characteristic, while the introduction of AEMA could provide more free amino groups for the conjugation of P3. The PEA based hydrogels and peptide functionalized hydrogel (Gel-g-P3) present multi-functions such as water uptake capacity, robust mechanical strength, enzymatic biodegradation, good hemocompatibility and cytocompatibility. Besides, the introduction of P3 made the Gel-g-P3 hydrogel more competitive in the coagulation caused by enhanced blood cell and platelet adhesion. Finally, PEA based hydrogels and Gel-g-P3 hydrogel was applied at a bleeding site and infected full-thickness wound. The Gel-g-P3 hydrogel showed better performance in hemostasis and wound healing. All these results demonstrated the peptide-functionalized PEA based hydrogel might be promising candidates for hemorrhage control and wound repairing.

4. MATERIALS AND METHODS Materials. L-lysine monohydrochloride (99%), p-toluenesulfonic acid monohydrate (TosOH·H2O, ≥98.5%), adipoyl chloride (98%), 1,4-butanediol (98%) and p-nitrophenol (98%) were used without further purification. Toluene, ethyl acetate, acetone, N, N’-dimethylacetamide (DMAc), N, N’-dimethylacetamide (DMF), diethyl ether, dichloromethane (DCM), dimethyl sulfoxide (DMSO), trifluoroacetic acid (TFA) and isopropanol were purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd., and purified by calcium hydride before use. Triethylamine (TEA, ≥99.0%) was purchased from Sinopharm Chemical Reagent Co., Ltd., and dried via refluxing with calcium hydride. Methoxy polyethylene glycol (mPEG, molecular weight (Mw) of 34


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2000), methacryloyl chloride (MAC), and methacrylic anhydride (MA) were purchased from Sinopharm Chemical Reagent Co., Ltd. 2-aminoethyl methacrylate hydrochloride (AEMA) was obtained from InnoChem Science & Technology Co., Ltd. Fmoc-Arg(Pbf)-OH, Fmoc-Phe-OH, Fmoc-Lys(BOC)-OH, Fmoc-Asp-OtBu, Fmoc-Gly-OH, HOBt (anhydrous), HBTU and N, N’-diisopropyl (DIEA) were purchased from GL Biochem (Shanghai) Ltd. Trypsin (Type IX-S, from porcine pancreas, lyophilized power, 13 000-20 000 BAEE units per mg protein) was purchased from Sigma-Aldrich Co., Ltd. Fluorescein isothiocyanate (mixture of 5- and 6isomers) was purchased from Tokyo Chemical Industry Co., Ltd. Synthesis of 4-Lys-4-MA and mPEG-MA Polymers. The synthesis route of Lys-4 and di-p-nitrophenyl adipate (NA) monomer was shown in Figure S1 (Supporting Information) according to the previous studies.30, 50 As excess NA can make the reaction cross-linked in static condition, it is important to control the feed ratio of NA and Lys-4 (molar ratio ≤ 1:1) and stirring ratio to obtain the water soluble polymer.29 In brief, 1.00 g Lys-4 and 0.51 g NA were dissolved in 5 mL DMAc at 80 oC. After being dissolved completely, 3.20 g TEA was added, meanwhile the solution was kept stirring (200 rpm) at 80 oC for overnight (Scheme 1a). The solution was then precipitated in ethyl acetate and the resulting yellow solids were washed and extracted by Soxhlet extractor for 24-48 h. The products were dried in vacuum at 80 oC for 24 h to obtain the 4-Lys-4 polymer. 2.00 g 4-Lys-4 was dissolved in 10 mL deionized (DI) water at room temperature. The pH value of the 4-Lys-4 solution was then adjusted to 8-9, and 1.30 g MA was added dropwise and reacted at 0 oC for overnight. The resulting solution was dialyzed for several days (dialysis membrane cutoff Mw of 500) and then lyophilized in vacuo at -50 oC 35



for 48 h to obtain the 4-Lys-4-MA polymer. mPEG-MA was synthesized as described in the previous study.12 As shown in Scheme 1b, 10 g mPEG was dried in vacuum and then dissolved in DCM, and excess MAC was added to the mPEG solution and reacted at 0 oC. The resulting solution was precipitated in diethyl ether and dried in vacuo at room temperature to get mPEG-MA. Yields of 4-Lys-4, 4-Lys-4-MA and mPEG-MA polymers were 84.4%, 76.7% and 95.3%, respectively. The molecular weight of 4-Lys-4 was investigated by Gel permeation chromatography (GPC) using THF as eluent (Waters-2690D equipped with Styragel columns). Synthesis of RGDK, RRRFK and RRRFRGDK Peptides. The three types of peptides RGDK (P1), RRRFK (P2) and RRRFRGDK (P3) were synthesized by the solid phase peptide synthesis method using peptide synthesizer.51 Taking RGDK for example, before reaction, 2.00 g 2-chlorotrityl chloride resin (loading 1.2 mmol/g) was first immersed in 20 mL DMF for 30 min. After draining off the DMF, Fmoc-Lys(BOC)-OH (4.8 mmol) and DIEA (7.2 mmol) in 20 mL DMF were added and reacted at room temperature for 1.5 h. Then, the resin was washed with 20 mL DMF for 3 times after removing the reaction solution. Subsequently, Fmoc deprotection was carried out with piperidine in DMF (20% v/v) at room temperature for 30 min and after that the resin was washed again with 20 mL DMF for 3 times. The presence of free amino groups was checked by ninhydrin solution (1% v/v in methyl alcohol). Thereafter, Fmoc-Gly-OH (4.8 mmol), HOBT (7.2 mmol), HBTU (7.2 mmol) and DIEA (7.2 mmol) in 20 mL DMF were added and reacted at room temperature for 1.5 h. Then the steps were repeated for several times to finish all the sequences. After the peptide synthesis was completed, the resin was washed with DMF and DCM for 3 times and dried in vacuum for 24 h. The peptide was cleaved from the resin, and the 36


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side chain protecting groups were removed using TFA/DMF/TIS (95:2.5:2.5 v/v/v) for 2 h. After washing using TFA for 2 times, the cleavage mixture were collected, concentrated and precipitated in ether. Finally, the precipitate was dissolved in DI water and then freeze-dried in vacuo for 48 h. Yields of P1, P2 and P3 were 83.5%, 87.2% and 80.1%, respectively. Formation of Hydrogels. To prepare the 4-Lys-4-MA hydrogel, 0.20 g 4-Lys-4-MA polymer was dissolved in 0.9 mL DMSO and 0.1 mL DI water and then 20 mg APS was added. After dissolving, the above solution was placed under a UV lamp (365 nm, 100 W) for 30 min to form the 4-Lys-4-MA hydrogel. For other hydrogels, the feed amount of 4-Lys-4-MA, mPEG-MA and AEMA was summarized in Table 1. To facilitate RRRFRGDK (P3) to conjugate onto the surface of the Gel-2 hydrogel, 0.5 g P3 was first dissolved in 10 mL DI water, then 0.09 g EDC and 0.55 g NHS were added to activate the carboxyl groups (Scheme 1c).52 The pH of solution was adjusted to 6.5, and the Gel-2 hydrogel was immersed in the solution overnight. Finally, the resulting hydrogel (designated as Gel-g-P3) was carefully washed with DI water for several times to remove the excessive P3. FITC-labeled P3 was used to determine the conjugation density of Gel-g-P3.24 The fluorescence intensity of the surfaces was acquired using a spectro fluorophotometer (SHIMADZU RF-600) and the conjugation density on hydrogel surface was calculated. Table 1. The feed ratio of hydrogels. 4-Lys-4-MA Code (g) 4-Lys-4-MA 0.20 4-Lys-4-MA/AEMA 0.15 Gel-1 0.09 Gel-2 0.11

mPEG-MA (g) 0.06 0.04

AEMA (g) 0.05 0.05 0.05

37


DMSO (mL) 0.90 0.90 0.90 0.90

DI water (mL) 0.10 0.10 0.10 0.10


Gel-3

0.13

0.02

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0.05

0.90

0.10

Hydrogel Morphology. The morphologies of the hydrogels were characterized by scanning electron microscopy (SEM, HITACHI, TM3000). Before observation, each hydrogel was immersed in DI water at room temperature until it reached its swelling equilibrium. After that, the hydrogel was freeze-dried for 48 h at -50 oC. The obtained samples were stuck to a holder and sputter-coated with gold for the SEM observation.12 Swelling Ratio. The swelling ratio of the hydrogels was studied by calculating the gravimetric change. Each freeze-dried hydrogel with known weight (Wd) was immersed in 10 mL PBS (pH 7.4, 0.1 M) at 37 oC. The hydrogels were taken out every 10 min and the excessive water on surface was gently cleaned by a filter paper.12 After that, the hydrogels were weighed (Ws). The swelling ratio is calculated by Equation 1. Swelling ratio =

Ws ― Wd Wd

× 100%

(1)

Enzymatic Biodegradation. The biodegradation properties of hydrogels were studied by calculating the weight loss. In brief, the hydrogels with a known weight (W0) were immersed in 20 mL PBS (pH 7.4, 0.1 M) with typsin (0.15 mg/mL) and sodium azide (0.2 mg/mL), and then shaken at 37 oC. The hydrogels were taken out at the pre-determined time and washed with PBS for three times, and then freeze-dried at -50 oC for 48 h. The freeze-dried the hydrogels were weighed (Wt). The weight loss of the hydrogels was calculated by Equation 2. Weight loss (%) =

W0 ― Wt W0

× 100%

(2)

Mechanical Characterization. Before test, the swollen hydrogels were cut into a uniform 38


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size (a diameter of 15 mm and height of 10 mm for compressive test; a diameter of 8 mm and height of 10 mm for rheological test). The compressive modulus of hydrogels was measured in a “controlled force” mode by a DMA Q800 Dynamic Mechanical Analyzer (TA Instruments) with the maximum force of 0.01 N and the rate of 0.02 N/min. The compressive modulus is calculated according to the initial stress (σ) and strain (ε, 0.05-0.25%) using Equation 3. Compressive modulus =

σ2 ― σ1 ε2 ― ε1

(3)

The rheological test was performed by a TA rheometer (ARES). The experiment was conducted under a constant strain of 1% and the shear rate was varied from 0.1 rad/s to 100 rad/s at room temperature.14, 15 Hemolysis Activity of Peptides and Hydrogels. The erythrocytes from fresh mouse blood were obtained by centrifuging (1000 rpm, 10 min) and washed by Tris-HCl for several times. The erythrocytes were diluted to a concentration of 5% (v/v). 50 µL peptide solutions or hydrogel powder suspensions with different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50, 100 and 200 µg/mL for peptides; 625, 1250, 2500 and 5000 µg/mL for hydrogels) and 50 µL erythrocytes were mixed and shaken at 37 oC for 1 h. After that, the suspensions were centrifuged again for 10 min (1000 rpm), and the supernatant was transferred to a new 96-well plate. The absorbance of the solutions at 540 nm was read by a microplate spectrophotometer. 0.1% Triton X-100 served as the positive control and Tris-HCl served as the negative control. the hemolysis ratio of peptides or hydrogels was calculated using Equation 4. Hemolysis ratio (%) =

Ap ― Ab At ― Ab

× 100%

(4)

Cytotoxicity Test. The L929 Fibroblast cells were cultured and harvested as described in 39



previous studies.12,

13

The cytotoxicity on L929 Fibroblast cells was evaluated by MTS assay

according to the protocol as a function of concentration for peptides (50, 100, 150 and 200 μg/mL) after 24 and 48 h. Besides, the live/dead cell staining method was utilized to evaluate the cell viability after 24 h. Before staining, the supernatant was removed and the cells were rinsed twice with PBS. Then enough working solution (5 μL PI and 5 μL AM in 10 mL PBS) was added to the cells and cultured for 45 min. The working solution was removed and 10 μL fresh PBS was added for fluorescence microscopy observation. The cell viability of the hydrogels was also evaluated by MTS assay. Before test, the hydrogels were cut into a uniform size to fill the 24-well cell culture plate and sterilized as described in the previous study.12 Subsequently, the hydrogels were placed into the wells of the plate, and L929 Fibroblast cells (30 000 cells per well) were seeded and incubated for 24 and 48 h, respectively. The cytotoxicity was processed according to the previous studies and TCP was used as control. The cell viability was calculated by the OD value at 490 nm. The growth and proliferation of L929 Fibroblast cells incubated with hydrogels was studied by optical microscope as well as live/dead cell staining method after 24 h. Antibacterial Activity Evaluation. The antibacterial properties of peptides (P1, P2 and P3) were investigated as a function of concentration against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli). Firstly, the MIC value of peptides was evaluated. Briefly, the peptides were diluted to a concentration of 200, 100, 50, 25, 12.5, 6.25, 3.125 μg/mL. No peptide group and no bacteria group were set as positive control and negative control, respectively. Bacterial growth was determined by OD at 600 nm. The MIC was defined as the lowest peptide 40


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concentration in which no bacterial were grown at 37 oC after 18 h. To measure the OD at 600 nm, the bacterial suspensions were diluted to the OD 600 value at 0.24 ± 0.01. Then the peptides were added at a concentration of 50, 100, 150 and 200 μg/mL, and the suspensions were shaken at 150 rpm at 37 oC. The absorbance of bacterial suspensions (200 μL) at 600 nm was measured at a pre-determined time. Besides, the suspension was diluted with PBS for different times, and 1 mL bacterial solution was spread onto Luria-Bertani (LB) agar plates and incubated at 37 oC for 24 h to form viable colony units. The log reduction was calculated as Equation 5. log reduction = log A - log B

(5)

In this Equation, A represents bacteria colony units in the control group, and B represents bacteria colony units in the sample group. The condition of bacteria treated with P3 (50, 100, 150 and 200 μg/mL) after 8 h was further studied by the live/dead analysis. In brief, propidiumiodide (PI) and SYBR Green (SG) dye (10 mg/mL) were added to the bacteria suspension for 15 min. Then the stained bacterial suspension was centrifuged, washed and diluted with PBS for fluorescence microscopy observation. Finally, the untreated bacteria and bacteria treated with P3 (200 μg/mL) after 4 and 8 h was collected for SEM test. The bacteria were fixed using 2 wt% of paraformaldehyde for 2 h and then washed with distilled water. Antibacterial activities of hydrogels were performed by agar plate diffusion test against S. aureus and E. coli as described in the previous study.12 The zones of inhibition (ZOI) for hydrogels are estimated using Equation 6:

41



ZOI =

D ― d 2

Page 42 of 54

(6)

In this equation, D is the average of the outer diameter zone of the inhibition (mm) and d is the diameter of hydrogel (mm). Whole Blood Clotting of Hydrogels. The whole blood clotting of hydrogels (4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3) were evaluated and gauze was used as control.10, 12 In detail, 0.4 mL whole mouse blood with 0.04 mL sodium citrate solution (38 mg/mL) was added to cover the prewarmed hydrogels. Then 30 μL calcium chloride solution (0.2 M) was added for coagulation. The process was kept at 37 oC and 10 mL DI water was slowly mixed after 5 and 10 min, respectively. 10 mL solution was collected and centrifuged, and the obtained supernatant along with 20 mL fresh DI water were transferred and kept at 37 oC for 1 hour. Finally, the relative absorbance of samples and at 540 nm was measured (A) and the absorbance of 0.4 mL citrated whole blood mixed with 30 mL DI water was also measured (B) The blood clotting index (BCI)was calculated using Equation 7. BCI (%) =

A B

× 100%

(7)

Blood Cell and Platelet Adhesion Test. The hydrogels (4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3) were first prewarmed in PBS 37 oC for 1 h. The whole blood was then dropwise added to cover the hydrogels at 37 oC for 5 min. Platelet-rich plasma (PRP) was obtained by centrifuging the whole blood and the obtained PRP was dropwise introduced to cover the hydrogels at 37 oC for 1 h. Then, the hydrogels were washed by PBS for several times, fixed by 2 wt% of paraformaldehyde for 2 h and dehydrated by ethanol solution.10, 53 The dried hydrogels were observed by SEM. 42


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In vivo Hemostasis Study. A hemorrhaging liver mouse model was employed to investigate the in vivo hemostatic potential of the hydrogels (4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3). The hydrogels with the swelling ratio reaching to 90% were aaplied in the hemostasis experiment. The procedures were referred to the previous studies.12, 54-56 Briefly, the anesthetized mouse was fixed on a corkboard and its liver was exposed by abdominal incision. The corkboard tilted at about 30o and bleeding of the liver was induced by a 20 G needle. The hydrogel was immediately applied to the bleeding sites. The filter paper was changed every 15 s and the hemostasis time was recorded. Evaluation of Wound Healing. The wound healing performance of samples (P3 soaked gauze, 4-Lys-4-MA, 4-Lys-4-MA/AEMA, Gel-2 and Gel-g-P3) was investigated in a mouse model (C57BL/6, 22-25 g, male). The only gauze was also used as control. These mice were first anesthetized and their dorsal areas were depilated and cleaned. The full-thickness circular wounds with a diameter of 10 mm were created and then injected with S. aureus suspension (50 μL, 1.0 × 107 CFU/mL) for 1 h. After that, samples were utilized to cover the wounds. The samples were changed every 2 days. At least 3 mice were used for each group. The wound healing properties were evaluated by optical photographs and wound size reduction calculated as Equation 8: Wound area closure (%) =

A0 ― At A0

× 100%

(8)

Where A0 is the initial wound area and At is the wound area on the indicated day. For histological analysis, skins including the entire wound and adjacent normal skin were harvested and then fixed in 4% buffered paraformaldehyde after 15 days. The skins were 43



embedded in paraffin and stained with hematoxylin and eosin (H&E). Besides, blood was also collected after 15 days for enzyme-linked immunosorbent assay (ELISA, Neobioscience, Shenzhen China). Animal Care and Treatment. The mice (C57BL/6 mouse, 22-25 g, 6 weeks, male) used in this study were purchased from SLRC Laboratory Animal, Shanghai, China. The mice were maintained in a controlled environment with a 12:12 L/D cycle at 24 ± 2 oC and 60 ± 5% humidity. The experiments were approved and done in accordance with protocols approved by the experimental animal center of Tongji University, Shanghai, China. All care and handling of the animals were performed with the approval of Institutional Authority for Laboratory Animal Care. Statistical Analysis. All data were evaluated as mean ± standard deviation based on at least three repeated tests. Statistical significance is calculated using Student’s t-test or one-way analysis of variance (ANOVA).

ASSOCIATED CONTENT Supporting Information Synthesis of Lys-4 and NA (Figure S1); 1H NMR spectra of mPEG-MA (Figure S2); the chromatogram and MS spectra of P1, P2 and P3 (Figure S3-S5, Table S1-S3); FT-IR spectra of P1, P2 and P3 (Figure S6); pore size of hydrogels (Figure S7); calculation results of crosslinking density of hydrogels (Table S4); biodegradation of hydrogels in PBS buffer (Figure S8); the comparison of hemolysis ratio of the peptides in this study with other literatures (Figure S9); 44


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MICs of peptides (Table S5); the OD value at 600 nm of untreated E. coli and S. aureus (Figure S10, S11); Total blood loss from the damaged livers (Figure S12); A list of clotting time and blood loss of commercial hemostatic materials (Table S6); The bacteria separated from wound tissues (Figure S13) (PDF)

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS The research is supported by the National Key Research and Development Program of China (project number: 2017YFB0309001) and the Natural Science Foundation of Shanghai (18ZR1400400, 18ZR1400500).

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TOC Peptide-Functionalized Amino Acid Derived Pseudoprotein Based Hydrogel with Hemorrhage Control and Antibacterial Activity for Wound Healing

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Peptide-Functionalized Amino Acid Derived Pseudoprotein Based

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