Fabrication of biodegradable multi-layered nanosheets to reinforce the bursting strength and maintain high adhesiveness for a wound dressing in partial hepatectomy

摘要

Introduction: We have proposed free-standing biodegradable nanosheets composed of poly (L-lactic acid) (PLLA) for biomedical applications. The nanosheets with a thickness of less than 100 nm exhibit unique properties such as high adhesiveness and exquisite flexibility. In fact, they can be adhered to moist organs without any adhesive agents. Recently, we demonstrated that the nanosheets acted as an effective wound dressing in a gastric operations in mice. However, they burst easily due to their nano-thickness. In this paper, we propose PLLA nanosheets with multi-layer structure inside. We expected the high adhesiveness of the nanosheet would be maintained by controlling each layers to nano-thickness, and its resistance to bursting would be reinforced by the multi-layer structure. Moreover, we evaluated whether the multi-layered nanosheets act as hemostats in partial hepatectomy, which is an appropriate in vivo system to test both functions (the resistance to bursting and the adhesiveness). Materials and Methods: The mult-layering of sodium alginate (Na-Alg, 20 mg/mL) and PLLA (10 mg/mL) was repeated 3-10 times by multiple rounds of spin-coating (4,000 rpm, 20 s) on a SiO_2 substrate. The substrate was immersed in an aqueous solution of 20 mg/mL CaCl_2 to allow gelation of the Na-Alg layers between PLLA nanosheets. The composite film was released from the substrate into water. Only two sides of the film was fusion-cut sealed with a Poly sealer~® (Fuji Impulse, Japan) at 187°C. The sealed film was incubated in EDTA to remove alginate layers. After washing with water, the multi-layered nanosheets were obtained. The bursting pressure and adhesive strength of the nanosheets were measured by a micro-scratch tester and a custom-made bursting tester. In a partial hepatectomy rat model, the 5-layered nanosheet supported with PVA film was sealed on the incised liver (size: 10 mm). Blood loss just after the sealing was measured for 5 min. As negative and positive controls, only PVA film and clinically-used TachoSil~® were tested. Results and Discussion: The mult-layered nanosheets were fabricated by simple combination procedures: (ⅰ) multi-layering of PLLA and alginate, (ⅱ) gelation of the alginate layers, (ⅲ) fusion-cut sealing and (ⅳ) elution of alginate layers. The multi-layered nanosheet was very flexible structure, which appeared whitish due to internal multilayers. Moreover, they exhibited an effective configuration to reinforce the bursting strength in parallel with the number of layers, and their adhesive strength were maintained at a high level as expected. In fact, the multi-layered nanosheets were found to be a potential to act as a novel wound dressing for hemostats (Fig. 2, blood loss of 5-layered nanosheet, only PVA film, and TachoSil: 0.65±0.12,2.60±0.20, and 0.94±0.17 g, respectively), and they tended to show reduced tissue adhesion. Conclusion: The free-standing multi-layered nanosheet composed of PLLA would be an ideal candidate for an alternative to conventional therapy in hepatectomy and other operations accompanied by bleeding.