Synthesis and Properties of Hemostatic and Bacteria-Responsive in

May 9, 2016 - Immediate hemorrhage control and infection prevention are pivotal for saving lives in critical situations such as battlefields, natural ...
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Synthesis and Properties of Hemostatic and Bacteria-Responsive in Situ Hydrogels for Emergency Treatment in Critical Situations Yazhong Bu,†,§ Licheng Zhang,‡,§ Jianheng Liu,‡ Lihai Zhang,‡ Tongtong Li,‡ Hong Shen,† Xing Wang,† Fei Yang,*,† Peifu Tang,*,‡ and Decheng Wu*,† †

Beijing National Laboratory for Molecular Sciences, State Key Laboratory of Polymer Physics & Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China ‡ Department of Orthopaedics, Chinese PLA General Hospital, Beijing 100853, China ABSTRACT: Immediate hemorrhage control and infection prevention are pivotal for saving lives in critical situations such as battlefields, natural disasters, traffic accidents, and so on. In situ hydrogels are promising candidates, but their mechanical strength is often not strong enough for use in critical situations. In this study, we constructed three hydrogels with different amounts of Schiff-base moieties from 4-arm-PEGNH2, 4-arm-PEG-NHS, and 4-arm-PEG-CHO in which vancomycin was incorporated as an antimicrobial agent. The hydrogels possess porous structures, excellent mechanical strength, and high swelling ratio. The cytotoxicity studies indicated that the composite hydrogel systems possess good biocompatibility. The Schiff bases incorporated improve the adhesiveness and endow the hydrogels with bacteria-sensitivity. The in vivo hemostatic and antimicrobial experiments on rabbits and pigs demonstrated that the hydrogels are able to aid in rapid hemorrhage control and infection prevention. In summary, vancomycin-loaded hydrogels may be excellent candidates as hemostatic and antibacterial materials for first aid treatment of the wounded in critical situations. KEYWORDS: PEG, hydrogels, hemostasis, antibacteria, pH-responsive

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the wounds often have irregular shapes, these films or sheets may not fit the wounds very well.19 On the contrary, in situ cross-linking hydrogels are widely explored as promising materials in urgent hemostasis because of their fitness to wounds with various shapes, the advantage of simple drug formulation, and the ability to deliver both hydrophilic and hydrophobic drugs.20−22 Peng et al. used polyethylene glycol (PEG) and poly(allylamine hydrochloride) (PAA) to fabricate in situ forming hydrogels for hemorrhage control.23 A chondroitin sulfate-PEG in situ adhesive hydrogel was also developed with potential applications in wound healing and regenerative medicine.24 Because of the rough movement of the wounded during their transport to safe places and the need of assistant pressure with gauzes when large areas of hemorrhage occur, these materials applied in urgent hemostasis must be tough enough to resist being broken. However, the mechanical properties of the in situ hydrogels are usually too poor to satisfy the requirements of critical situations. Polyethylene glycol (PEG) is a very popular material which is used extensively in scaffolds for tissue engineering applications such as articular cartilage, neural, and bladder tissue regeneration which is essentially nonimmunogenic, antifouling, and nontoxic.25,26 Recently, Sakai et al. designed a 4-arm-PEG

INTRODUCTION A large number of wounded personnel can come into being in a very short time in critical situations such as battlefields, natural disasters, traffic accidents, and so on. In these situations, uncontrollable bleeding and infections pose the most significant fatality risks, by which more than half of deaths are caused.1−3 Due to the fact that one-third of prehospital deaths originate from hemorrhage in emergency civilian settings, immediate control of hemorrhage is essential for lowering fatality rates as bleed outs can occur within 5−10 min.4−6 Another key step is to prevent infection of the wounded because the wounds are easily infected in dirty environments, impeding the healing process and even causing life threatening complications.7−9 Since immediate professional treatment of the wounds is rarely possible in many extreme cases, simple but effective first aid of hemorrhage control and infection prevention plays a vital role to save the lives of patients before they have access to professional treatment. Many efforts have been tried to achieve effective hemorrhage control and infection prevention.10−15 Hoekstra et al. designed hydrocolloid dressings which were more efficient than the traditional gauze in the wound treatment.16 Krishnan et al. made freeze-dried sheets of polymerized fibrin which supported hemostasis and wound healing.17 Then the fibrin sheets were used to load tetracycline for hemostasis and antibacteria.18 Those materials generally need to be made in the form of thin films or sheets in advance. However, in critical situations where © XXXX American Chemical Society

Received: March 16, 2016 Accepted: May 9, 2016

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DOI: 10.1021/acsami.6b03235 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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Scheme 1. Vancomycin-Loaded Hydrogel Systems Formed by (A) 4-Arm-PEG-NH2 Reacting with (B) 4-Arm-PEG-NHS, (C) 4Arm-PEG-CHO, and (D) Polymer Networks Composed of Different Molar Ratios of A: B: C, Their Adhesion to Tissues and Bacteria-Responsive Release of Vancomycin

hydrogel by combining two symmetrical 4-arm-PEGs.27 The 4arm-PEG hydrogel has high mechanical strength with a maximum breaking strength of 9.6 MPa because of its homogeneous structure, which is tough enough to be used as the hemostasis material in critical situations. There are many ways to prevent infections of the wounds,28,29 a simple treatment is to load antibiotics in the wound dressings.7 Vancomycin is a first-generation glycopeptide antibiotic which has already been proven to own sustained microbiologic inhibitory activity.30,31 Its antibacterial mechanism is to inhibit the synthesis of the bacterial cell wall, which has no cross-resistance to other antibiotics.32 It rarely generates resistant strains and is effective for most Gram-positive bacteria.33,34 So vancomycin is a preferred choice in criticalsituation medicine.35 The release of antibiotics in materials is favorable to be responsive to bacteria, which can restrict the rise of drug resistant bacteria and decrease the undesirable cytotoxicity effects.36 The change of pH is very common in the environment with fast growth of bacteria, motivating the development of pH-responsive antibacteria delivery systems.37 The Schiff base is pH-responsive and trends to be quickly hydrolyzed under a weakly acidic condition.38−40 Recently, Shi et al. reported a doxorubicin (DOX) loaded hydrogel based on Schiff base-conjugated DOX-chitosan as a pH sensitive drug delivery system for cancer treatment.41 Zhang et al. constructed a multiresponsive Schiff-base chitosan hydrogel for controlled release of bioactive molecules.42 Generally, the bacteria can produce lactic acid during their metabolic process, leading to local weak acidification,36 so Schiff base is suitable for constructing bacteria-sensitive antibiotics release system. In this study, we develop in situ hydrogels with excellent mechanical properties and pH-responsive release of vancomycin that yield good performance of hemorrhage control and infection prevention. We chose 4-arm-PEG to build welldefined hydrogel networks for yielding high mechanical strength, and Schiff base cross-links were tuned by adjusting ratios of 4-arm-PEG-NH2, 4-arm-PEG-NHS, and 4-arm-PEGCHO to optimize the mechanical properties and pH-responsive release behaviors (Scheme 1). The hydrogels can tightly adhere

to the tissues in situ through fast reaction between minor amine groups of tissue proteins and succinimide or formyl groups of 4-arm-PEG. Hydrogels with more Schiff-base moieties yielded better performance in antimicrobial experiments in vitro. Hemostatic and antimicrobial experiments in vivo using rabbits and pigs proved the hydrogels could achieve rapid hemorrhage control and infection prevention.

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EXPERIMENTAL SECTION

Materials. 4-Arm poly(ethylene glycol) (4-arm-PEG-OH, Mw = 10 kDa, Mw/Mn = 1.02), 4-arm poly(ethylene glycol) succinimidyl (4arm-PEG-NHS, M w = 10 kDa, Mw/Mn = 1.03) and 4-arm poly(ethylene glycol) amine (4-arm-PEG-NH2, Mw = 10 kDa, Mw/ M n = 1.03) were purchased from SINOPEG, China. 1-(3(Dimethylamino)propyl)-3-ethylcarbodiimide hydrochloride (EDCI), 4-carboxybenzaldehyde, and 4-dimethylaminopyridine (DMAP) were purchased from ENERGY CHEMICAL. All chemicals were used as received. All other chemicals were of analytical grade. Synthesis of 4-Arm Poly(ethylene glycol) aldehyde (4-armPEG-CHO). 4-Arm-PEG-CHO was prepared and characterized according to the process described by the previous report.42 Briefly, 4-carboxybenzaldehyde (150 mg, 10 equiv), EDCI (191.7 mg, 10 equiv), and DMAP (61.08 mg, 5 equiv) were dissolved in dry CH2Cl2 (40 mL), followed by the addition of 4-arm-PEG-OH (1 g, 1 equiv) under a nitrogen atmosphere. The system was stirred at 37 °C for 24 h after which the mixture was washed with 1 M HCl (3 × 40 mL), saturated NaHCO3 (3 × 40 mL), and brine (3 × 40 mL). The organic layer was then dried partially under reduced pressure and the polymer was obtained as a white solid after drying under vacuum. Yield: 90%. 1 H NMR (300 MHz, CDCl3): δ 3.25 (t, 8H; CH2), 3.64 (bs, ∼910H; OCH2CH2O), 3.85 (s, 8H; CH2CH2OCO), 4.61 (s, 8H; CH2OCO) 7.95 (m, 8H; ArH), 8.24 (m, 8H; ArH), 10.03 (m, 4H; CHO); 13 C NMR (300 MHz, CDCl3): δ 47.2, 64.6, 68.9, 70.6, 129.5, 130.4, 134.9, 139.0, 165.6, 191.8. Preparation of Hydrogels. In the process of preparing the hydrogels, the phosphate buffer (pH 7.4) was used to dissolve the polymers. First of all, precursor solution 1 was prepared by dissolving 4-arm-PEG-NH2 in a sample bottle, while three kinds of precursor solution 2 were prepared by dissolving 4-arm-PEG-NHS and 4-armPEG-CHO in another sample bottle with the ratios of 4-arm-PEGNHS: 4-arm-PEG-CHO to be 1:0, 0.7:0.3 and 0.5:0.5. The preparation steps were performed at room temperature. Then, by using a dual syringe, the same volume of precursor solution 1 and 2 were simultaneously injected into the molds to obtain hydrogels. According B

DOI: 10.1021/acsami.6b03235 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces to the precursor solution 2 used, Gels 1−3 were prepared with the ratios of 4-arm-PEG-NH2: 4-arm-PEG-NHS: 4-arm-PEG-CHO to be 1:1:0, 1:0.7:0.3, and 1:0.5:0.5. The polymer concentration was fixed at 15 wt % unless specially mentioned. Measurement of Gelation Time. When used on the wounds, the time that makes sense is when the hydrogels stop flowing and form physical barriers. So the gelation time was determined by the vial tilting method.43 We prepared the hydrogels in sample vials. The time at which there was no flow upon inverting the vial was regarded as the gelation time. Fourier Transform Infrared (FTIR) Spectra. Gels 2 and 3 were prepared according to methods described above and then freeze-dried at −50 °C for 24 h. The pure 4-arm-PEG-CHO was used as the control sample. Fourier transform infrared (FTIR) spectra of the dried samples were obtained using TENSOR-27 spectrometer (Bruker, German) in the frequency range 4000−400 cm−1 at a resolution of 2 cm−1 with a total of 32 scans by the potassium bromide tableting technique. Scanning Electron Microscopy (SEM). Gels 1−3 were prepared according to methods described above and then freeze-dried at −50 °C for 24 h. Then the dried samples were carefully stuck onto the conducting resin with double-sided adhesive and were sputter-coated with a thin layer of Pt for 120 s to make the sample conductive before testing. Field emission scanning electron microscopy (SEM) images were obtained at acceleration voltage of 5 kV on a JSM-6700F microscope (JEOL, Japan). Rheological Studies. Rheometry was performed on a Thermo Haake with a plate geometry (35 mm diameter) at 25 °C. The hydrogel discs (diameter = 35 mm, thickness = 1.2 mm) were tested at a gap distance of 1 mm. Before the tests, an amplitude sweep was first performed in order to define the linear viscoelastic region (LVR) in which the storage modulus is independent to the strain amplitude. Then an angular frequency ω of 15 rad s−1 and a deformation amplitude γ0 of 0.5% were selected to perform the rheological studies. Compression Testing. Gels 1−3 were prepared according to methods described above in cylindroid molds (15 mm in diameter and 7.5 mm in height). The compression testing was performed using a universal tensile machine (3365 Instron, U.S.A.). The dimensions of each sample were measured using a digital caliper before testing. The rate of compression testing were set at 1 mm/min until the sample fractured. Every kind of hydrogels were tested for 3 times. Swelling Ratio. The swelling ratio measurements were performed in the phosphate buffer (pH 7.4) according to the previous report.44 The discs of Gels 1−3 (15 mm in diameter and 4 mm in height) were dried at 25 °C for 24 h in an oven. The dried samples (∼0.1 g) were weighted (Wd) and then immersed into 50 mL of phosphate buffer at 37 °C. At specific intervals of time, the samples were taken out and weighed (Ws). The swelling ratio is calculated as Swelling ratio (%) = (Ws − Wd)/Wd × 100%. Cytotoxicity Studies. Cell viability was measured using quantitative MTT cytotoxicity assay and was assessed by contacting extracts of the hydrogels. 3T3 mouse fibroblasts were suspended in cell culture medium and seeded into 96-well microculture plates with a density of 1 × 104 cells/100 μL/well and incubated for 24 h at 37 °C in a 5% CO2 humidified incubator to obtain a monolayer of cells. Cell medium was replaced with hydrogel extracts or cell culture medium containing various controls (i.e., Gels 1−3 with and without vancomycin) and further incubated for an additional time (24 or 72 h). The sample solution was removed and the cells were incubated with 50 μL of 1 mg/mL of MTT in PBS for 2 h. Finally, the PBS solution was replaced with 100 μL of DMSO to dissolve formazan, and the absorbance of the DMSO solution was detected at 570 nm (reference 650 nm). The relative cell viability was calculated as the ratio between the mean absorbance value of the sample and that of cells cultured in the medium. Samples with relative cell viability less than 70% were deemed to be cytotoxic. For each sample, 5 independent cultures were prepared and cytotoxicity test was repeated 3 times for each culture. In Vitro Drug Release Study. We built a model to simulate the release of hydrogels used as the hemostatic and antimicrobial materials

on the skin. Vancomycin was encapsulated into the hydrogel matrix in situ to produce drug-loaded hydrogels. When covered on the skin, only the sides of hydrogels adhesive to skin released vancomycin. As a result, the hydrogels used for release were formed in the container with the diameter of 10 mm and height of 2 mm. Release studies were performed in buffer solutions with different pH (7.4 and 5) in which the vessels containing hydrogels were put. The collected buffer solutions were tested by HPLC at 230 nm to determine vancomycin contents. Torsion Experiments. To evaluate the adhesion of the hydrogels, we investigated their adherence to pigskin. Pigskin was selected due to its biological similarity to human dermis.45 The hydrogels were formed in situ on the surface of the skin by using a dual syringe. Then torsion stress was applied on the hydrogels to test their adherence flexibility on the skin. Bursting Pressure Test. Bursting pressure test was performed according to methods described by previous methods described by Azuma et al.46 The pig abdominal aorta vein was used in this experiment. The vein was linked to a syringe pump and filled with PBS solution. A 3 mm hole was made on the vein surface after which the hydrogels were formed in situ on the puncture site. The bursting pressure was measured 5 min after the forming of the hydrogels. The pressure at which it began to decrease was considered the bursting pressure. All measurements were repeated six times. In Vitro Antimicrobial Assays. To evaluate the antimicrobial activity of the hydrogels, Gram-positive bacteria S. aureus was used as the model microorganism. Hydrogels for the antibacterial assays were prepared in 96-well tissue culture plates (TCP). 100 μL of hydrogels with different concentrations of vancomycin were formed in 96-well tissue culture plates at 37 °C. Then 200 μL S. aureus solution with 1 × 106 CFU/mL which had been grown in tryptic soy broth (TSB, BD) overnight in an incubator at 37 °C was introduced into the wells. The optical density readings of microorganism solutions were monitored by measuring OD 600 nm after 24 h. Antimicrobial activities have been further tested through a spread plate method.47 Hydrogels containing the vancomycin of 250 μg/mL were challenged with S. aureus solution with the concentration of 1 × 106 CFU/mL. At predetermined times, microbial suspensions were withdraw and diluted sequentially, and then plated on 1.5% LB agar plates. The plates were incubated for 24 h at 37 °C. Microbial colonies were formed and counted. The S. aureus in the wells were used as the control. The results are expressed as surviving fractions (%) = survivor count of the sample/cell count of the control ×100%. The experiments were repeated for 5 times. In Vivo Antimicrobial Assays. The DNA of the S. aureus (RN6390-GFP) was modified to make sure that green fluorescent protein was expressed during the growth process of the bacteria. Rabbits of 2−4 months old were used in the experiment. Two traumas with the diameter of 2 cm were made on both sides of the backbone of the rabbits after which 200 μL RN6390-GFP solution (1 × 108 CFU/ mL) was introduced onto the wounds. The rabbits were raised in the cage for 24 h before the infection formed. Then, 2 mL of hydrogels containing vancomycin (250 μg/mL) were formed in situ on the wounds. Three days later, the hydrogels were removed, and the skin of the rabbits was incised for laser confocal microscopy. Gauzes and hydrogels without vancomycin and with vancomycin were used as the controls. Rabbit Liver and Pig Skin Laceration. New Zealand white rabbits of 2−4 months old and Bama miniature pigs of 2 months were used in the experiment. In the rabbit liver laceration experiment, an incision of 1 cm in length and 0.5 cm in depth was made with a surgical scalpel on the liver. Two mL of the hydrogels were formed in situ on the wound, and the incision was inspected visually. Compression with gauzes alone served as the control. Immediately following the surgery, the liver was excised, fixed in formalin for 3 days, placed in a histological cassette, and then dehydrated in graded ethanol solutions. Following fixation and dehydration, the sample was embedded in paraffin, and sections of cut into 3−5 μm were stained with hematoxylin and eosin. Three rabbits were used in this study (n = 3). In the pigskin laceration experiment, a cylinder wound with 3 cm in C

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Figure 1. Characterization of hydrogel precursors and hydrogels. (A) 1H and (B) 13C NMR spectra of 4-arm-PEG-CHO. (C) FTIR spectra of 4arm-PEG-CHO, Gel 2, and Gel 3. The characteristic peaks at 1702 cm−1 referred to CO stretching of aldehyde peaks. (D) The gel time of various hydrogels (10 and 15 wt %). (E) SEM images (bar scale =10 μm), (F) swelling ratios, (G) compression testing, and (H) rheological analysis (tan δ = loss modulus/storage modulus) of Gels 1−3 (15 wt %). diameter and 1 cm in depth was made. Then 2 mL of the hydrogels were formed in situ on the incision, and the incision was inspected visually (n = 3). The gauze was used as the control (n = 3). Three pigs were used in the experiment. Rabbit Femoral Artery Hemostasis Tests. Rabbits of 2 months were used in the femoral artery hemostasis tests. The femoral arteries of rabbits were peeled from the surrounding tissues and later clipped by the surgical scissors. Hemostatic forceps were then used to clamp the fractured blood vessels to stop the blood flowing out of the vessels. Two mL of hydrogels were applied to the bleeding site by the dual injector. After 30 s of gelation, hemostatic forceps were removed and the bleeding site was inspected visually to find out whether the bleeding was stopped or not. Two surgeries were performed on each animal, and three rabbits were used in this experiment.

The hydrogels can be easily formed at room temperature by simply mixing these three compositions of 4-arm-PEG-NH2, 4arm-PEG-NHS, and 4-arm-PEG-CHO with different ratios using the dual syringe. This simple operation is very easy to handle for the wounded or ambulance men in the critical situations where any time-consuming operation might lead to death. The rate of Schiff base reaction between 4-arm-PEGNH2 and 4-arm-PEG-CHO is slower than that of amidation reaction between 4-arm-PEG-NH2 and 4-arm-PEG-NHS. To testify formation of the Schiff base in the hydrogel network, we characterized the structure of the hydrogels containing Schiff bases (Gel 2 and Gel 3) as well as the original reagent, 4-armPEG-CHO. As shown in Figure 1C, the disappeared characteristic peaks at 1702 cm−1 representing CO stretching of aldehyde peaks after the forming of the Gel 2 and Gel 3 proved the formation of Schiff bases in the hydrogel networks. To quickly stop bleeding, it is greatly preferred to form the hydrogels in situ on the trauma as fast as possible, so we first optimized the gelation time by adjusting the solid content. We investigated the gelation time using 10 and 15 wt % of the solid contents. All the gel times were more than 1.5 min for these three hydrogels obtained from 10 wt % of the solid content (Figure 1D), too slow to match the requirement of fast hemostasis. Increasing the solid content to 15 wt % significantly reduced the gel times to around 20−25 s. It is preferred that the physical barriers to blood loss form as fast as possible to prevent extra bleeding. So we utilized the 4-arm-PEG with the concentration of 15 wt % to fabricate the hydrogels in the next experiments.

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RESULTS AND DISCUSSION Synthesis and Characterization of Hydrogels. We incorporate the Schiff-base moieties into the cross-linked network to make the hydrogels pH-sensitive. To improve stability of the hydrogels, we adopted aromatic Schiff bases which are more stable than the aliphatic counterparts because of the conjugation.42 So, we produced the benzaldehydefunctionalized PEG (4-arm-PEG-CHO) by esterification of 4arm-PEG-OH with 4-carboxybenzaldehyde. The peaks of aldehyde (10.03 ppm), benzene ring (8.24, 7.95, 129.5, and 130.4 ppm), and ester methylene (4.51 ppm) were clearly observed from 1H and 13C NMR spectra of the 4-arm-PEGCHO (Figure 1A and Figure 1B). The results manifested the successful preparation of the well-defined 4-arm-PEG-CHO polymer. D

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ACS Applied Materials & Interfaces The porous structures of the hydrogels used as hemostatic agents are very important in stopping bleeding since they can absorb the exudates of the wounds, thus increasing the concentration of the red blood cells and plates.5 Besides, the porous structures can maintain a suitable moisture environment for the wounds, beneficial for effective wound healing.13,48 Figure 1E affirmed the formation of highly porous structures of the three hydrogels. The pore sizes were 8.4 ± 3.8, 6.5 ± 2.7, and 7.4 ± 3.5 μm for Gels 1−3, respectively, calculated by the Image-pro Plu. Using well-defined 4-arm-PEGs with very narrow polydispersity (1.02−1.03), the pores are uniform and the incorporation of Schiff bases does not affect the uniformity. The resultant hydrogels with uniform structures may be promising in the hemostasis. Because of the porous structure and the hydrophilicity of PEG, the hydrogels are supposed to be able to absorb a large amount of water from serum to concentrate red blood cells and plates. Figure 1F showed the swelling ratios of the Gels 1−3 were 1550%, 1610%, and 1790%, which were larger than many other polysaccharide dressings.49−51 In this sense, the PEG hydrogels with high swelling ratios meet with the requirement for effective absorption of water from serum to concentrate coagulation factors and cells. High mechanical strength is also very necessary for the hydrogels to avoid their crush during given assistant pressure to help stop bleeding and the violent movement of the wounded. So we investigated the mechanical strength of the hydrogels by the compression tests, and the results were summarized in Figure 1G. The maximum breaking stress of the Gels 1−3 were 4.94, 2.16, and 0.89 MPa. When used on the wounds, the hydrogels could stand with stress greatly larger than the normal arterial blood pressure of the healthy adult (0.016 MPa). Meanwhile, the outside pressure which is tens or hundreds of times the normal arterial blood pressure could be applied on the hydrogels for assistant hemostasis. Figure 1G also showed that incorporating Schiff-base moieties into the network weakened the mechanical strength, in accordance with the tan δ in the rheological studies (Figure 1H). The tan δ that equals the ratio of loss modulus to storage modulus represents the intensity of the network.52−54 Compared with the amido bond, Schiff base is a kind of weaker chemical bond, leading to weaker mechanical strengths of Gels 2 and 3. However, the hydrogels with Schiff bases are still tough enough for utilization in critical situations. Cytotoxicity Studies. A critical objective in the development of hemorrhage control and bacteria-resistant tissuecontacting dressings is to keep their functions without compromising their ability to support the adhesion and proliferation of tissue cells.36 The materials used in this study are PEG and vancomycin. It is well-known that PEG has good biocompatibility and vancomycin is a kind of antibiotic that has been used in clinic. To clearly illustrate the cytotoxicity of the materials, we used 3T3 mouse fibroblasts to assess the biocompatibility of the various hydrogels coatings. The 3T3 mouse fibroblasts were cultured with exacts from Gels 1−3 with and without vancomycin for 1 and 3 days. As depicted in Figure 2A, compared with the control group, the cell viability remained over 80% when the concentrations of the hydrogels were up to 10 mg/mL. The results demonstrated the hydrogels have low cytotoxicity to the 3T3 mouse fibroblasts. Figure 2B indicated that encapsulation of the vancomycin did not increase the cytotoxicity: i.e., still over 90% cell viability was detected

Figure 2. Cytotoxicity of (A) the hydrogels and (B) the vancomycinloaded hydrogels in NIH 3T3 fibroblasts after incubation for 1 and 3 days. (Values were presented as mean ± standard deviation, n = 5).

even when concentration of loaded vancomycin was up to 15 mg/mL. Tissue Adhesion. The materials used in the hemostasis as sealing agents are needed to be able to adhere to the tissues and bear the pressure of the bleeding. To test the adhesion, the three hydrogels were formed in situ on the surface of the pigskin and despite the stress applied, the hydrogels could adhere to the skin and did not fall off the skin (Figure 3A). To further test their ability to adhere to the skin when bleeding happens, we used the bursting pressure tests (Figure 3B), and the results are summarized in Figure 3C. The bursting pressure of Gel 1 (116.5 ± 4.4 mmHg) was slightly lower than the arterial blood pressure (120 mmHg), but Gel 2 (164.7 ± 21.9 mmHg) and Gel 3 (197.6 ± 49.8 mmHg) showed higher values. For Gel 1, it covalently adheres to tissue through amide bonds,24 whereas for Gel 2 and Gel 3, except for the covalent linkage of amide bonds and Schiff bases between tissues and hydrogels, there also exist the π−π stacking interactions between the benzene ring of the aromatic Schiff bases in the network and the protein of the tissues.55 So incorporation of Schiff bases into the hydrogels improves the adhesion ability of the hydrogels to withstand pressure greater than normal arterial blood pressure and makes them better sealing agents. In Vitro Drug Release Study and Antimicrobial Assays. The release experiments were carried in PBS buffer solutions at 37 °C. To optimize the antibacterial effect of the loaded vancomycin, it is favorable that vancomycin can be responsively released according to the growth of bacteria. The E

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Figure 4. (A) Schematic of the release experiment. (B) The release profiles of the hydrogels in pH 5.0 and 7.4. (Values were presented as mean ± standard deviation, n = 3).

Figure 3. Tissue adhesion experiments results. (A) Photographs of the hydrogels adhered to skin tissues, under torsion in which rhodamine B (red) was incorporated for better observation. (B) Schematic and results (C) of the bursting pressure tests.

Schiff bases can be cleaved to aldehyde and amino under the weakly acid environment. Since the bacteria produce lactic acid during their metabolic activity to cause acidification of the microenvironment,36 the Schiff bases are suitable linkers for the bacteria-responsive hydrogels. To evaluate the pH-responsive release of vancomycin, we used the release medium with the pH of 5 to simulate the weakly acidic environment where the bacteria reproduced. In the situations where the hydrogels were used, only one side of the hydrogels would contact the release medium. So the hydrogels for vancomycin release were put in containers with the diameter of 10 mm and height of 2 mm and only one side was accessible to the release medium (Figure 4A). HPLC was adopted to monitor the components of the release medium. Figure 4B affirmed that at pH 7.4, very little vancomycin was released from the hydrogels (16% for Gel 1, 21% for Gel 2, and 28% for Gel 3). However, while at pH 5, the release amount increased obviously for the hydrogels containing Schiff bases (78% for Gel 2 and 93% for Gel 3) and more Schiff bases meant faster release speed. The results suggested that incorporation of Schiff bases into the hydrogels made the release of vancomycin responsive to pH, thus bacteria-sensitive hydrogels are created. The results also indicated that when exposure to bacteria, vancomycin-loaded hydrogels containing more Schiff base moieties may have better performance on elimination of the bacteria. To further prove that, we conducted in vitro antibacterial tests. The S. aureus were first challenged with the three kinds of hydrogels loading different amount of vancomycin in TCP for 24 h and the microbial proliferation was assessed by optical density readings of microorganism solutions at 600 nm. Although the PEG hydrogels could reduce the initial attachment of the bacteria,56 they did not kill or inhibit the growth of the bacteria. As a result, these hydrogels without vancomycin did not perform well in the antibacterial experiments (Figure 5A). The loading

Figure 5. (A) The amount of S. aureus when challenged with the hydrogels under various concentrations of vancomycin compared with the amount in plates. (B) Time course of surviving S. aureus challenged with the hydrogels when the concentration of vancomycin was 250 μg/mL.

of vancomycin in the hydrogels facilitated antibacterial ability and with the same amount of vancomycin loaded, the hydrogels containing more Schiff-base moieties eliminated the bacteria F

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Figure 6. Procedure and laser confocal microscopy results of the in vivo antimicrobial assays (scale bar = 100 μm). The green bright dots represent viable RN6390-GFP.

wounds and can be very promising as antibacterial agents in critical situations. Hemostasis Experiments. Besides evaluating the in vivo antibacterial ability, we also investigated the hemostatic effect of the hydrogels on significant bleeding using laceration models of the rabbit liver and pigskin to simulate the severe trauma. Just as in the in vivo antimicrobial assays, Gel 2 was also applied in the hemostasis experiments. In the rabbit liver model, a surgical scalpel was used to induce bleeding that would not be stopped by compression (Figure 7A). When Gel 2 was placed on the wounds as a physical barrier, it quickly stopped the blood loss, and the complete hemostasis of the trauma was visually observed (Figure 7B). To further explore the possible hemostasis mechanism of the hydrogels, the organ was washed

more easily. The antibacterial activity of the three hydrogels was further evaluated by analyzing the survival rate of bacteria upon contact with the three hydrogels at various exposure times with the spread plate method. Figure 5B affirmed that the most fast bacteria elimination speed is Gel 3, and the slowest is Gel 1. These results clearly disclose that the incorporation of Schiffbase moieties into the network can make the hydrogels more efficient in the antimicrobial activity and more Schiff bases mean better antibacterial effects. In Vivo Antimicrobial Assays. To further demonstrate the antibacterial capacity of the vancomycin-loaded hydrogels, we used rabbits as the model for antimicrobial assays in vivo (Figure 6). The infection models were constructed on wounds of rabbit skin with diameters of 2 cm after injection of bacteria RN6390-GFP for 24 h. Here, Gel 2 with suitable mechanical strength, adhesion, and release rate was applied on the infection model. Laser confocal microscopy was used to detect bacteria on the wounds 3 days later. The antibacterial properties of gauzes, vancomycin solution, Gel 2 without vancomycin, and vancomycin-loaded Gel 2 were investigated in the antibacterial experiment (Figure 6). The green bright dots in Figure 6 represent viable RN6390-GFP. We set the treatment time of materials at 3 days because it often takes 2 to 3 days for the patients to be accessible to the professional treatment. Figure 6 showed there were numerous viable bacteria observed on the traumas covered by gauzes, vancomycin solution, and Gel 2 without vancomycin. It was surprising that when vancomycin solution was used on the wounds, the amount of bacteria were almost the same as that of the gauzes group. We think this happened because the vancomycin solution used might be easily washed away by the exudates of the wounds. Gel 2 had a better performance than gauzes and the vancomycin solution because of the antifouling properties of the PEG,57 but many viable bacteria still existed. Notably, on Gel 2 with vancomycin coated surface, no bacteria were observed by laser confocal microscopy. The results robustly illustrate that the hydrogels with vancomycin can inhibit the growth of bacteria on the

Figure 7. Photographs taken during the liver laceration model. (A) A wound with a length of 1 cm, (B) Hydrogels formed in situ on the trauma, (C,D) H&E stained micrographs, showing significant accumulation of red blood cells and plates within the incision site (scale bars = 100 μm). G

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ACS Applied Materials & Interfaces

Figure 8. (A) Photographs taken during the pigskin laceration animal model: (a) stripping by scalpel, (b) hydrogels applied trauma which showed complete hemostasis immediately, (c) gauzes applied trauma which was still bleeding even after 5 min, and (d) a Bama miniature pig of 2 months used in this experiment. (B) Photographs taken during the femoral artery hemostasis tests (the inconspicuous femoral artery was highlighted by the yellow circle and ellipse): (a) the femoral artery, (b) clipping by scissors and hemostasis by forceps, (c) Gel 2 applied to the wound, (d) the femoral artery surrounded by Gel 2, and (e) complete hemostasis achieved after removing the hemostatic forceps.

situ on the bleeding site by the injector (Figure 8B-c), and the vessels were completely surrounded by the hydrogels (Figure 8B-d). After 30 s, the hemostatic forceps were removed and no bleeding was observed (Figure 8B-e) because the hydrogel obstructed the fractured blood vessels as a sealant. The results definitely demonstrate the potential of the hydrogels in stopping bleeding of the leaky vessels, which cannot be achieved by traditional gauzes.

and stained using the H&E method. The staining results showed significant cell accumulation in the trauma (circled by dashed lines in Figure 7C and D). We deduced that the accumulation was partly caused by the water-absorption of hydrogels from serum. So we concluded that when Gel 2 was applied on the wounds, it first acted as a physical barrier to blood loss. Then, the water was absorbed from the serum by the hydrogels to help concentrate the blood coagulation factor, red blood cells, and plates, thus fast hemostasis was achieved. In the pigskin laceration model, a hole with the diameter of 3 cm and a depth of 1 cm was dug using the scalpel (Figure 8A-a). There was no more bleeding coming out from the wounds after Gel 2 was applied on the trauma for 30 s (Figure 8A-b). In comparison, the control group using gauzes could not stop bleeding even after 5 min (Figure 8A-c). The results from laceration models of the rabbit liver and pigskin indicate that the hydrogels possess excellent hemostatic effect on the severe traumas. Vascular injuries also need to be considered, as there is a high rate of occult vascular damage in critical situations.58 So besides evaluation of the hemostatic effect on severe traumas, the capability of sealing the broken artery was also investigated by rabbit femoral artery hemostasis tests. As shown in Figure 8B-a, the femoral arteries of rabbits were first peeled from the surrounding tissues and then clipped by the surgical scissors. Large areas of hemorrhage happened. Just as the clinical treatment, we first used hemostatic forceps to clamp the fractured blood vessels to stop the blood flowing out of the vessels (Figure 8B-b). Then 2 mL of hydrogels were formed in

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CONCLUSIONS In this study, three kinds of vancomycin-loaded hydrogels with different amounts of Schiff-base moieties were constructed from 4-arm-PEG-NH2, 4-arm-PEG-NHS, and 4-arm-PEGCHO. The hydrogels exhibit porous structures, excellent mechanical strength, and high swelling ratio, which are favorable for hemostasis in critical situations. The cytological assay indicated that the composite hydrogel systems possess good cell biocompatibility. The introduction of Schiff bases into the network improves the adhesiveness and endows the hydrogels bacteria-sensitivity. Hemostatic and antimicrobial experiments in vivo using rabbits and pigs displayed that the hydrogels are able to aid in rapid hemorrhage control and infection prevention. The work demonstrates that the composite hydrogel systems may be wonderful candidates for the first aid treatment of the wounded in critical situations.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (F.Y.). H

DOI: 10.1021/acsami.6b03235 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces *E-mail: [email protected] (P.T.). *E-mail: [email protected] (D.W.).

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Author Contributions §

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to MOST (2014CB932200 and 2014BAI11B04), NSFC (21504096, 51573195, 21174147, and 21474115), and the “Young Thousand Talents Program” for financial support.

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