Effect of alkyl chain length on the interfacial strength of surgical sealants composed of hydrophobically-modified Alaska-pollock-derived gelatins and poly(ethylene)glycol-based four-armed crosslinker

Colloids and Surfaces B: Biointerfaces 146 (2016) 212–220

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Effect of alkyl chain length on the interfacial strength of surgical sealants composed of hydrophobically-modified Alaska-pollock-derived gelatins and poly(ethylene)glycol-based four-armed crosslinker Ryo Mizuta a,b , Temmei Ito a,b , Tetsushi Taguchi a,b,∗ a

Graduate School of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8577, Japan Polymeric Biomaterials Group, Biomaterials Field, Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan b

a r t i c l e

i n f o

Article history: Received 25 January 2016 Received in revised form 8 June 2016 Accepted 10 June 2016 Available online 11 June 2016 Keywords: Sealant Alaska-pollock-derived gelatin Hydrophobically-modified gelatin Poly(ethylene)glycol Adhesion

a b s t r a c t Surgical sealants are widely used clinically. Fibrin sealant is a commonly used sealant, but is ineffective under wet conditions during surgery. In this study, we developed surgical sealants composed of hydrophobically modified Alaska-pollock-derived gelatins (hm-ApGltns) with different alkyl chain lengths from C3 to C18 and a poly(ethylene)glycol-based 4-armed crosslinker (4S-PEG). The burst strength of the hm-ApGltns-based sealant was evaluated using a fresh porcine blood vessel and was found to increase with increasing alkyl chain length from 167 ± 22 to 299 ± 43 mmHg when the substitution ratio of amino groups of ApGltn was around 10 mol%. The maximum burst strength was observed when stearoyl-group modified ApGltn (Ste-ApGltn)/4S-PEG-based sealant was used, displaying 3-fold higher burst strength than the original ApGltn (Org-ApGltn)/4S-PEG sealant, and 10-fold higher than the commercial fibrin sealant. Ste-ApGltn/4S-PEG-based sealant was biodegraded in rat subcutaneous tissue within 8 weeks without severe inflammation. By molecular interaction analysis using surface plasmon resonance, the binding constant of Ste-ApGltn to fibronectin was found to be 9-fold higher than that of Org-ApGltn. Therefore, the developed sealant, in particular the Ste-ApGltn/4S-PEG-based sealant, has potential applications in the field of cardiovascular surgery as well as thoracic surgery. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Surgical sealants are widely used clinically for the treatment of pulmonary air leaks and anastomotic sites between living tissues. One commonly used sealant is fibrin sealant, and its components are biopolymers including fibrin and thrombin [1]. The sealing mechanism of this fibrin is based on human blood coagulation. Fibrin sealant has excellent biocompatibility and versatility; however, it does not possess sufficient sealing effect because of its low interfacial bonding strength to tissues. Therefore, the molecular design of a surgical sealant is required that will adhere to living tissue and organs under wet conditions during surgery.

∗ Corresponding author at: Polymeric Biomaterials Group, Biomaterials Field, Research Center for Functional Materials, National Institute for Materials Science, 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan. E-mail address: [email protected] (T. Taguchi). http://dx.doi.org/10.1016/j.colsurfb.2016.06.017 0927-7765/© 2016 Elsevier B.V. All rights reserved.

Recently, a synthetic urethane-based sealant was developed for hemostasis of cardiovascular anastomosis [2]. It is composed of a viscous diisocyanate prepolymer, and forms urethane bonds between the isocyanate groups and amino groups in proteins. Urethane-based sealants have rapid curing characteristics and high bonding strength to soft tissues in the presence of water. In addition, they display load following capability because of their high elasticity. However, the polymerized product is non-absorbable and thus mediates adverse effects such as inflammation, infection and calcification [3]. Research into bio-inspired materials as surgical sealants has highlighted mussel-inspired adhesives as attractive candidates for sealing under wet conditions. It is known that the L-3, 4dihydroxyphenylalanine in mussel adhesive proteins plays an important role in the adhesion of mussels in aquatic environments [4–7]. Nanomaterials have also been extensively studied as potential surgical sealants. Fujie et al. reported that a nanosheet composed of chitosan and alginate showed burst strength similar to fib-

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rin sealant, along with high biocompatibility and biodegradability [8–10]. Furthermore, adhesion using silica or iron oxide nanoparticles reveals nanobridging that achieves rapid and strong closure properties [11,12]. However, these nanomaterials have not yet demonstrated sufficient sealing effects for clinical applications. In our previous study, we revealed that tissue adhesives containing hydrophobically-modified porcine-derived gelatins have excellent bonding strength onto fresh vascular media compared with non-modified gelatin [13–17]. However, high concentration porcine-derived gelatin solutions show low fluidity at room temperature because of their high content of imino acids, such as proline and hydroxyproline, and the need for heat treatment before sealant use. Gelatin is one of the most popular biopolymers used in biomedical applications owing to its biodegradability and biocompatibility. Although porcine- and bovine-derived gelatin are most commonly used, in this study fish gelatin was employed owing to its unique properties [18]. The gelatin of cold-water fish in particular (e.g., cod, hake, Alaska pollock, and salmon) shows higher fluidity compared with porcine- or bovine-derived gelatin [19]. Therefore, we have chosen Alaska-pollock-derived gelatins (ApGltn) instead of porcine-derived gelatin as a base material for surgical sealants [20], because it has a low transition temperature owing to its low content of imino acids [19,21,22]. We synthesized various hydrophobically-modified ApGltns (hm-ApGltn) with different hydrophobic groups and introduction ratios and evaluated their sealing effects on wet tissues by combining Hm-ApGltns with a poly(ethylene)glycol-based 4-armed crosslinker [23].

2. Materials and methods 2.1. Materials

Fig. 1. Chemical structure of the components of sealants. (a) Preparation and structure of hm-ApGltn. (b) Chemical structure of pentaerythritol poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-PEG).

a)

110

-CH3 strech (methyl: 2879cm-1)

-CH2 -strech (methylene: 2935cm-1)

3

Transmittance(%)

2

Alaska-pollock-derived gelatin (ApGltn) was kindly donated by Nitta Gelatin Inc. (Osaka, Japan). Propanoyl chloride (Pro:C3), hexanoyl chloride (Hx:C6), lauroyl chloride (Lau:C12) and stearoyl chloride (Ste:C18) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ethanol (EtOH), ethyl acetate (EtOAc), dimethylsulfoxide (DMSO), triethylamine (TEA), 2,4,6-trinitrobenzensulfonic acid (TNBS), sodium dodecyl sulfate (SDS), 6 mol/L hydrochloric acid (6N-HCl), pyrene and acetone were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Pentaerythritol poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-PEG, Sunbright® PTE-100GS, MW = 10,000) was purchased from NOF Corporation (Tokyo, Japan). A porcine blood vessel was purchased from Funakoshi Corporation (Tokyo, Japan). Saline was purchased from Otsuka Pharmaceutical Co., Ltd. (Tokyo, Japan). Collagenase was purchased from Nacalai Tesque Inc. (Kyoto, Japan).

100 Org

90

Pro-apGltn Hx-apGltn

80 70

3500

3300

3100

Ste-apGltn

2900

2700

2500

Wavenumber(cm-1)

b)

2.2. Synthesis and characterization of hm-ApGltn Modification of ApGltn with hydrophobic groups was carried out by a nucleophilic substitution reaction of the amino group of ApGltn with fatty acid chlorides (hydrocarbon chain length: C3–18) in DMSO (Fig. 1a) [13–17]. In brief, ApGltn (30 g) was first dissolved in DMSO (742.5 ml) at a concentration of 4 wt% at 37 ◦ C. Fatty acid chlorides were then added into the ApGltn/DMSO solution at different concentrations from 10 to 40 mol% to change each theoretical introduction ratio. After stirring for 30 min, TEA (7.5 ml) was added to start the chemical reaction. The mixture was stirred for 15 h at room temperature in a N2 atmosphere, hm-ApGltn was then precipitated in a mixture of cold ethanol and ethyl acetate. The precipitate was washed three times in cold ethanol (3 l) to remove any remaining fatty acid chlorides, DMSO and TEA. Hm-ApGltn was

Lau-apGltn

N-H strech (secondary amine: 3276cm-1)

Ste-apGltn Lau-apGltn Hx-apGltn Pro-apGltn Org

80

60

40

20

0

δ/ppm Fig. 2. Characterization of hm-ApGltn. (a) FTIR spectra of hm-ApGltn with different alkyl chain lengths. (b) 13 C NMR of hm-ApGltn with different alkyl chain lengths. The FTIR and 13 C NMR measurements were performed using Org-ApGltn, 6.4Pro-ApGltn, 8.5Hx-ApGltn, 9.0Lau-ApGltn and 9.6Ste-ApGltn.

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Table 1 Characterization of hydrophobically-modified ApGltns. Abbreviation

Fatty acid chloride added (mol%)

Hydrophobic group modification (mol%)

Org-ApGltn 3.2Pro-ApGltn 6.4Pro-ApGltn 13.8Pro-ApGltn 2.1Hx-ApGltn 8.5Hx-ApGltn 18.3Hx-ApGltn 3.8Lau-ApGltn 9.0Lau-ApGltn 19.0Lau-ApGltn 4.0Ste-ApGltn 9.6Ste-ApGltn 18.7Ste-ApGltn

– 10 20 40 10 20 40 10 20 40 10 20 40

– 3.2 6.4 13.8 2.1 8.5 18.3 3.8 9.0 19.0 4.0 9.6 18.7

Tg (◦ C) 12.7 14.7 14.3 15.0 14.8 14.9 16.3 14.2 14.5 15.6 15.0 15.2 14.1

± ± ± ± ± ± ± ± ± ± ± ± ±

0.9 0.6 0.6 0.7 0.4 0.4 0.4 0.1 0.8 0.4 0.7 1.0 0.4

CMC (g/L)

Yield (%)

10.5 9.8 9.8 7.9 8.2 8.0 7.4 8.5 7.9 7.6 8.5 7.6 6.9

– 88.3 96.4 91.1 87.0 90.7 85.6 83.6 88.6 95.5 96.5 92.5 95.7

CMC, critical micelle concentration; Tg , glass-transition temperature. Tg data represent the mean ± SD of three samples.

obtained by drying under reduced pressure after filtration of the precipitate. The modification ratio of the hydrophobic groups was calculated by determination of the residual amino group using trinitrobenzenesulfonic acid [24]. First, hm-ApGltn or Org-ApGltn (10 mg) were dissolved in DMSO (10 ml) to prepare a 0.1% ApGltn/DMSO solution. By adding 0.1% TEA/DMSO and 0.1% TNBS/DMSO into the ApGltn solution, TNBS reacted with the residual amino groups of ApGltn. TNBS exhibits a UV absorbance at 340 nm so the residual amino groups were determined using a microplate reader (GENios A-5082, Tecan Japan, Kanagawa, Japan). While the glasstransition temperature (Tg ) was measured using a differential scanning calorimeter (DSC8230, Rigaku, Tokyo, Japan). The critical micelle concentration was determined by the fluorescent intensity of pyrene. Furthermore, the introduction of hydrophobic groups into the amino groups of ApGltn was confirmed using Fourier transform infrared spectroscopy (FTIR) (FTIR-8400S, Shimadzu Co., Ltd., Kyoto, Japan) and 13 C-nuclear magnetic resonance (13 C NMR, AL300, JEOL, Tokyo, Japan). 2.3. Evaluation of the rheological properties of the hm-ApGltn-based sealant The rheological properties of the hm-ApGltn-based sealant were determined using a rheometer (MCR301, Anton Paar GmbH, Granz, Austria). The storage modulus was obtained by frequency dependent measurements (angular frequency, 0.1–100 (1/s); strain, 5%), and the shear strength was determined from the result of storage modulus at a frequency of 1.0 (1/s) [25–27]. Fibrin sealant was used as a control material.

Fig. 3. Burst strengths of hm-ApGltn/4S-PEG-based sealant after application on a porcine blood vessel. (a) Burst strength of hm-ApGltn/4S-PEG with different modification ratios and alkyl chain lengths. (b) The effect of alkyl chain length on burst strength. Data represent the mean ± SD of five samples. *p < 0.05, **p > 0.05.

2.4. Swelling behavior of the hm-ApGltn-based sealant

2.5. Measurement of the burst strength of a porcine blood vessel

Disc-shaped sealant (diameter, 4.0 mm; thickness, 0.5 mm) was prepared by mixing hm-ApGltn solution (40 wt%, 0.1 M phosphatebuffered saline (PBS) pH 8.0) and 4S-PEG solution (0.1 M PBS pH 8.0). The disc-shaped sealant was immersed in saline at 37 ◦ C, then the weight of the sealant (Ws ) was measured. The disk-shaped sealant was then immersed for 24 h in deionized water at 37 ◦ C to remove sodium ions. The weight of a disk-shaped sealant after freeze drying (Wd ) was also measured and the swelling ratio of the sealant was calculated using the following equation [28]:

The measurement of burst strength of a porcine blood vessel was performed according to a standard method (ASTM, F2392-04) [29]. A fresh porcine blood vessel was employed as the adherent. The tissue sample was prepared by inserting a pinhole (3 mm in diameter) in the center of the disk-shaped blood vessel. Sealant composed of hm-ApGltns and 4S-PEG of fixed diameter (15 mm) and thickness (1.0 mm) was applied to cover the hole. The burst strength was then measured by running a saline from the lower portion of the tissue at a flow rate of 2 ml/min at 37 ◦ C. After the sealant was fractured, the maximum burst strength was defined as the individual burst strength of the sealant. Fibrin sealant was used as a control. To analyze the adhesion mechanism, a cross section of the blood vessel after the burst strength measurements was observed [30]. The test sample was fixed in a 10% formalin neutral buffer solution,

Swelling ratio =

Ws − Wd Wd

where Ws and Wd are the weight of the swollen and dried sealant, respectively.

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stained with hematoxylin and eosin (H&E), and analyzed under an optical microscope (BX51, Olympus, Tokyo, Japan). 2.6. Enzymatic degradability of the hm-ApGltn/4S-PEG-based sealant Disc-shaped sealant (diameter, 4.0 mm; thickness, 1.0 mm) was prepared by mixing hm-ApGltn solution (40 wt%, 0.1 M PBS pH 8.0) and 4S-PEG solution (0.1 M PBS pH 8.0). To remove the effect of calcium formation by PBS in the sealant, the disk-shaped sealant was immersed in Tris-HCl buffer (pH 7.4) at least 4 h prior to use. First, collagenase was added to Tris-HCl buffer (pH 7.4, 2.5 mM CaCl2 ) at a concentration of 200 ␮g/ml, and the disk-shaped sealant was then immersed in the collagenase mixture (2 ml). The weight remaining (%) was calculated by measuring the weight after immersion compared with the weight before immersion [31–33] using the following equation: Weight remaining (%) =

Wt × 100 Wt0

where Wt and Wt0 are the sealant weights after and before immersion, respectively. 2.7. in vivo biodegradability of the hm-ApGltn/4S-PEG-based sealant Biodegradability of the hm-ApGltn/4S-PEG-based sealant was observed by implantation into the subcutaneous tissues of mice. Initially, the sterile hm-ApGltn solution (40 wt%, 0.1 M PBS pH 8.0) was prepared by UV radiation. The disk-shaped sealant was prepared using sterilized hm-ApGltn solution at a fixed diameter (5.0 mm) and thickness (0.5 mm). The disk-shaped sealant was then implanted onto the back of mice (ICR, 8-week-old females; Charles River Japan Inc.) for 1, 2, 4, and 8 weeks. After the incubation period, each tissue around the sealant was removed and fixed in 10% formalin neutral buffer solution. The test sample and the surrounding tissue was stained with H&E and then observed using an optical microscope (BX51, Olympus).

215

2.9. Statistical analysis Statistical analysis was carried out using Welch’s t-test. A value of p < 0.05 was considered to indicate statistical significance. The data were represented as the mean ± standard deviation (SD). 3. Results 3.1. Synthesis and characterization of hm-ApGltn Hm-ApGltns were successfully synthesized using a previously reported method [13–17]. Propanoyl chloride (Pro:C3), hexanoyl chloride (Hx:C6), lauroyl chloride (Lau:C12) and stearoyl chloride (Ste:C18) were used as fatty acid chlorides. By changing the initial concentrations of fatty acid chlorides, hm-ApGltns with three different modification ratios of hydrophobic groups were obtained. From the determination of residual amino groups, each modification ratio for the hydrophobic groups was calculated as shown in Table 1. By FTIR, the increased concentration of methyl stretch (2879 cm−1 ), methylene stretch (2935 cm−1 ) and secondary amine stretch (3276 cm−1 ) was observed. The intensity of each stretch was increased up to the alkyl chain length as shown in Fig. 2a. Furthermore, the intensity of methylene groups was also observed at around 40 ppm by 13 C NMR (Fig. 2b), indicating that hm-ApGltn had been successfully obtained. 3.2. Rheological properties of the hm-ApGltn/4S-PEG-based sealant Using the MCR 301 rheometer, the storage modulus and shear modulus of each sealant were measured as rheological properties. The storage modulus of the hm-ApGltn/4S-PEG-based sealant was higher than that of the commercial fibrin sealant as shown in Supplementary Fig. 1a. The shear modulus was determined from the value of the storage modulus at 1.0 (1/s). The slight increase in the shear modulus conferred by the introduction of hydrophobic groups was measured. As shown in Supplementary Fig. 1b, the shear modulus of the hm-ApGltn/4S-PEG-based sealant with a long alkyl chain length was higher than that of the hm-ApGltn/4S-PEGbased sealant with a short alkyl chain length.

2.8. SPR analysis

3.3. Swelling behavior of the hm-ApGltn/4S-PEG-based sealant

To determine the interaction between hm-ApGltn and fibronectin, surface plasmon resonance (SPR; Biacore X, GE Healthcare Japan, Tokyo, Japan) [34–36] was performed. Either hm-ApGltn or Org-ApGltn were immobilized onto the surface of the carboxymethyl dextran sensor chip (GE Healthcare Japan) using an amine coupling kit (BR-1000-50, GE Healthcare Japan). By flowing a mixture of N-hydroxysuccinimide and N-ethyl-N (3-dimethylaminopropyl) carbodiimide hydrochloride across the sensor chip, the carboxymethyl dextran on the surface was activated. Either hm-ApGltn or Org-ApGltn solution (2000 ␮g/ml, 10 mM acetic buffer pH 4.0) was injected for 7 min at a flow rate of 5 ␮l/min at 25 ◦ C, the excess active sites were then blocked using 1 M ethanolamine hydrochloride. After this coupling procedure, fibronectin solution (concentration: 25–200 ␮g/ml in Dulbecco’s PBS) was injected into the surface for 5 min at a flow rate of 20 ␮l/min at 25 ◦ C. Dulbecco’s PBS was used as running buffer for the whole procedure. The association constant (KA ) was calculated using the association rate constant (Ka ) and the dissociation rate constant (Kd ) as shown below:

The swelling ratio of the hm-ApGltn/4S-PEG-based sealant is shown in Supplementary Fig. 2. The water content of the ApGltn/4S-PEG-based sealant increased from about 80% to 90% after immersion for 240 min. The swelling behavior of the Ste-ApGltn/4S-PEG-based sealant was similar to that of the Org-ApGltn/4S-PEG-based sealant. After immersion for 240 min, the swelling ratio of the Org-ApGltn/4S-PEG-based sealant was 9.0 ± 0.1 compared with 8.4 ± 0.3 for the Ste-ApGltn/4S-PEG-based sealant. However, this difference was not significant. The swelling speed of the Ste-ApGltn/4S-PEG-based sealant was slower than that of the Org-ApGltn/4S-PEG-based sealant.

KA =

Ka Kd

3.4. Measurement of the burst strength The burst strength of the hm-ApGltn/4S-PEG-based sealants was measured according to the ASTM (F2392-04) method. The burst strengths of all hm-ApGltn/4S-PEG-based sealants were higher than that of the Org-ApGltn/4S-PEG-based sealant (Fig. 3a) and commercial fibrin sealant. In particular, the burst strength of the 9.6Ste-ApGltn/4S-PEG-based sealant was 10-fold higher than that of commercial fibrin sealant and 3-fold higher than that of OrgApGltn/4S-PEG-based sealant. In the case of short alkyl chain length, the modification ratio had little effect on burst strength,

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Fig. 4. Histology of hm-ApGltn/4S-PEG-based sealant after burst strength measurement. 6.4Pro-ApGltn, 8.5Hx-ApGltn, 9.0Lau-ApGltn and 9.6Ste-ApGltn were employed as hm-ApGltn. Org-ApGltn and fibrin sealant were used as control materials. The sealant (S) and tissue (T) are indicated in the picture.

whereas in the case of long alkyl chain length, the modification ratio was more important. Furthermore, the burst strengths of Lau and Ste-ApGltn/4S-PEG-based sealants were higher than those of Pro and Hx-ApGltn/4S-PEG-based sealants when the modification ratio was around 10 mol% (Fig. 3b). This indicated that the hm-ApGltn/4S-PEG-based sealant with long alkyl chain length effectively enhanced the burst strength. After measuring burst strength, cross-sections of the blood vessels were analyzed to assess the adhesion mechanism of the hm-ApGltn/4S-PEG-based sealants. As shown in Fig. 4, the hm-ApGltn/4S-PEG-based sealants remained bound to blood vessels even after burst strength measurements. Destruction of the sealants was observed in the cases of Pro or Hx-ApGltn/4S-PEGbased sealants. Tissue tearing was also observed (as shown in the upper surface view) indicating that the strong interfacial bond between Lau and Ste-ApGltn/4S-PEG-based sealants and blood vessels. In the case of Org-ApGltn/4S-PEG-based sealants and fibrin, adhesion failure was observed indicating a weak interaction between sealants and tissue. 3.5. Biodegradability of hm-ApGltn/4S-PEG-based sealants in vitro and in vivo To measure the biodegradability of hm-ApGltn/4S-PEG-based sealants, the disk-shaped sealants were immersed in collagenase solution. As shown in Fig. 5a, 9.6Ste-ApGltn/4S-PEG-based sealant was completely degraded within 5 h, compared with 3 h for Org-ApGltn/4S-PEG-based sealant. We also implanted these sealants onto the back of mice. As shown in Fig. 5b, 9.6SteApGltn/4S-PEG-based sealant was completely degraded within 8 weeks without severe inflammation. Cell infiltration into 9.6SteApGltn/4S-PEG-based sealant was also detected 4 weeks after implantation. Org-ApGltn/4S-PEG-based sealant was completely degraded within 4 weeks, whereas fibrin sealant was degraded within 2 weeks. 4. Discussion Synthesis of hm-ApGltn was performed by a nucleophilic substitution reaction of the amino group in the gelatin with fatty acid chlorides (hydrocarbon chain length: C3–18) in DMSO. Following determination of the residual amino groups using TNBS, hm-ApGltns with different alkyl chain lengths and modification

Table 2 Binding constants of hm-ApGltn to fibronectin obtained by SPR measurements.

Data are the average of three samples. *Significant difference (p < 0.05) among the constant values.

ratios were characterized using FTIR and 13 C NMR. The properties conferred by the introduced hydrophobic groups affected the critical micelle concentration (CMC) and the glass-transition temperature (Tg ). The decrease in CMC and Tg with increasing introduction ratio was caused by the aggregation of hydrophobic groups. These results also confirmed the successful modification of amino groups with hydrophobic groups. The high bulk modulus of surgical sealants was required for preventing oozing from anastomotic sites between living tissues or pulmonary air leaks [3]; therefore, the storage modulus and shear modulus of hm-ApGltn/4S-PEG-based sealants were characterized. Compared with the commercial fibrin sealant, the storage modulus of all hm-ApGltn/4S-PEG-based sealants was significantly higher. Furthermore, the shear modulus of the hm-ApGltn/4S-PEG-based sealants with long alkyl chains showed greater strength than those with short alkyl chains. This resulted from the formation of physical crosslinks by the hydrophobic groups introduced into the ApGltn molecules. This indicated that the bulk strength of surgical sealants was enhanced by combining hm-ApGltn and 4S-PEG. Also, the hmApGltn/4S-PEG-based sealants were formed by a simple stirring method. Rapid formulation is essential for use in surgical operations [3], indicating the potential application of these sealants clinically. The swelling behavior of sealants is another important property because excess swelling can cause sealants to spontaneously peel away from living tissues. As shown in Supplementary Fig. 2b, hm-ApGltn/4S-PEG-based sealants showed a low level of swelling compared with Org-ApGltn/4S-PEG-based sealants. This phenomenon was due to physical crosslinking by hydrophobic

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Fig. 5. Degradation behavior of Org-apGlrn/4S-PEG or 9.6Ste-ApGltn/4S-PEG-based sealants in vitro and in vivo. (a) In vitro enzymatic degradation behavior. (b) in vivo degradation behavior of Org-ApGltn/4S-PEG or 9.6Ste-ApGltn/4S-PEG-based sealant after implantation in mice subcutaneous tissue for 1, 2, 4 and 8 weeks. The sealant (S) is indicated in the picture.

groups of hm-ApGltns and indicated that hm-ApGltn/4S-PEGbased sealants had excellent characteristics for use in surgical operations. The burst strengths of all hm-ApGltn/4S-PEG-based sealants against blood vessels were higher than those of Org-ApGltn/4SPEG-based sealant (Fig. 3a). In particular, the burst strength of 9.6Ste-ApGltn/4S-PEG-based sealant was 10-fold higher than that of fibrin sealant and 3-fold higher than that of Org-ApGltn/4S-PEGbased sealant. This indicated that the introduction of hydrophobic groups, especially Ste groups, effectively enhanced the tissue penetration of sealants by the increasing the interfacial strength between tissue and sealant. Furthermore, the burst strengths of hm-ApGltn/4S-PEG-based sealants with long alkyl chains, including Lau and Ste groups, were higher than those with short alkyl

chains, such as Pro and Hx groups. This indicated that the bulk strength as well as interfacial bonding strength of sealants plays an important role in enhancing burst strength. Compared with each hydrocarbon chain, burst strength was increased with increasing alkyl chain length. This was due to the enhancement of interfacial strength between sealant and tissue by the promotion of interpenetration of hm-ApGltn with longer alkyl chains into tissue. We also evaluated the effect of modification ratios on burst strength. In the case of short alkyl chains, no significant difference was observed among the hm-ApGltns, whereas burst strength was increased up to a modification ratio of around 10 mol%, and then decreased (Fig. 3a), especially in the case of Ste-ApGltn/4SPEG-based sealant. This was due to the high hydrophobicity of Ste groups. The burst strength with a modification ratio of 10 mol%

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Fig. 6. Adhesion mechanism of hm-ApGltn/4S-PEG-based sealant on blood vessels.

was enhanced because of the hydrophobic interactions between sealant and tissue, while the decrease in burst strength of 18.7SteApGltn/4S-PEG-based sealant was because of the promotion of self-aggregation of Ste-ApGltn molecules by hydrophobic interactions. We also analyzed fracture patterns following burst strength measurements. In general, there are three kinds of fracture patterns: adhesion failure, cohesion failure and tissue tearing, with interfacial strength increasing in this order [30]. The fracture pattern after burst strength measurement of Org-ApGltn/4SPEG-based sealant was found to be a cohesion failure, whereas 9.6Ste-ApGltn/4S-PEG-based sealant still remained on the surface of the blood vessel even after burst strength measurement. These results correlated with the burst strength of the sealants, and indicated that the interfacial strength of 9.6Ste-ApGltn/4S-PEG-based sealant provided sufficient adhesive properties to the blood vessel. To investigate the enhanced adhesive behavior of hmApGltn/4S-PEG-based sealant to blood vessels, the interaction between hm-ApGltn and fibronectin was measured using SPR. Fibronectin is an extracellular protein expressed on blood vessels. Each Ste-ApGltn or Org-ApGltn was successfully fixed on the surface of the carboxymethyl dextran sensor chip by amine coupling.

After flowing fibronectin was applied to the surface of the sensor chips, the association constant (KA ) was calculated using the association rate constant (Ka ) and the dissociation rate constant (Kd ). The association constant (KA ) of 18.7Ste-ApGltn was about 25-fold higher than that of Org-ApGltn (Table 2). From these results, we proposed the sealing mechanism of hmApGltn/4S-PEG-based sealant as shown in Fig. 6. There are several types of extracellular matrix proteins expressed by blood vessels. One typical extracellular matrix protein is elastin, and the content of hydrophobic amino acids in elastin is about 96% [37]. We propose that the increased burst strength of hm-ApGltn/4S-PEG-based sealant is due to the enhanced interaction of hydrophobic groups with these hydrophobic amino acids. Other types of molecular recognition between fibronectin and collagen may also contribute to the enhancement of burst strength; for example, fibronectin contains a binding site for Gltn. In addition, the content of hydrophobic amino acids in fibronectin is about 50% [38]; therefore, the association between hm-ApGltns and fibronectin was high compared with Org-ApGltn as shown in Table 2. The in vitro and in vivo degradation behavior of 9.6SteApGltn/4S-PEG-based sealant was slow compared with OrgApGltn/4S-PEG-based sealant (Fig. 5). The delayed degradation of

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9.6Ste-ApGltn/4S-PEG-based sealant was due to the high crosslinking density of this sealant including both physical crosslinking and covalent crosslinking by 4S-PEG. Consequently, water replacement in 9.6Ste-ApGltn/4S-PEG-based sealant would be slow compared with Org-ApGltn/4S-PEG-based sealant. Cell infiltration into 9.6Ste-ApGltn/4S-PEG-based sealants was also observed 4 weeks after implantation. This indicated that 9.6Ste-ApGltn/4SPEG-based sealant acts as a scaffold for cell infiltration and proliferation. We previously reported that hydrophobic groups introduced into PEG at both terminal groups enhanced cellular spheroid formation [39]. The mechanism of spheroid formation involves the anchoring of hydrophobic groups into the phospholipid membrane of cells. Similarly, Ste groups in 9.6Ste-ApGltn can also anchor to phospholipid cell membranes to enhance cell infiltration into the sealant. In addition, 9.6Ste-apGlrn shows high binding to fibronectin, as shown in Table 2. Fibronectin is known as a cell adhesion protein [40], and possesses the RGD sequence (Arg-Gly-Asp sequence) to bind integrins on the cell surface [41,42]. Therefore, the 9.6Ste-ApGltn-fibronectin complex also works as a scaffold for cell infiltration promoting tissue regeneration. Furthermore, we have reported that hydrophobically modified gelatin-based materials showed high biocompatibility in an in vivo inflammation test [14], and exhibited cytocompatibility in an in vitro cell proliferation test [16]. Taken together, the sealants described in this study, and particularly 9.6Ste-ApGltn/4SPEG-based sealant, are good candidates to be applied for use in cardiovascular surgery, as well as thoracic surgery. 5. Conclusion A surgical sealant consisting of hm-ApGltn and 4S-PEG was prepared. Modification of ApGltn with hydrophobic groups effectively enhanced interfacial strength to porcine blood vessels even under wet conditions. Hm-ApGltn/4S-PEG-based sealants with long alkyl chains such as Lau and Ste showed high burst strength compared with short alkyl chains. The molecular interaction between hmApGltn and fibronectin contributed to this enhancement of burst strength. Also, hm-ApGltn/4S-PEG-based sealants showed enzymatic biodegradability without severe inflammation, and function as scaffolds for tissue integration in mice subcutaneous tissue. Therefore, our sealant has the potential to be applied clinically for cardiovascular surgery, as well as thoracic surgery. Acknowledgments We thank Mr. Y. Masuda, Ms. T. Kojima and Dr. K. Yoshizawa of the National Institute for Materials Science for their technical support. This work was financially supported in part by the Japan Society for the promotion of Science KAKENHI (Grant No. 16H04524). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2016.06. 017. References [1] Y.M. Bhat, S. Banerjee, B.A. Barth, S.S. Chauhan, K.T. Gottlieb, V. Konda, J.T. Maple, F.M. Murad, P.R. Pfau, D.K. Pleskow, U.D. Siddiqui, J.L. Tokar, A. Wang, S.A. Rodriguez, Tissue adhesives: cyanoacrylate glue and fibrin sealant, Gastrointest. Endosc. 78 (2013) 209–215. [2] M. Eto, S. Morita, M. Sugiura, T. Yoshimura, R. Tominaga, T. Matsuda, Elastomeric surgical sealant for hemostasis of cardiovascular anastomosis under full heparinization, Eur. J. Cardiothorac. Surg. 32 (2007) 730–734.

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