Preparing Silk Fibroin Nanoﬁbers through Electrospinning: Further Heparin Immobilization toward Hemocompatibility Improvement Marília Cestari, † Vinícius Muller, ‡ Jean Henrique da Silva Rodrigues, § Celso V. Nakamura, § Adley F. Rubira, † and Edvani C. Muniz* ,† † Departamento de Química, Universidade Estadual de Maringa ́ UEM, 87020-900 Maringa, Brazil ‡ Centro de Engenharias e Ciê ncias Exatas, Universidade Estadual do Oeste do Parana ́ UNIOESTE, 85903-000 Toledo, Brazil § Laborató rio de Inovaç ã o Tecnoló gica no Desenvolvimento de Fa ́ rmacos e Cosme ́ ticos, Departamento de Ciê ncias Ba ́ sicas da Saú de, Universidade Estadual de Maringa ́ UEM, Av. Colombo 5790 87020-900 Maringa ́ , Parana ́ , Brazil ABSTRACT: Sodium heparin (HS) was immobilized on the surface of the silk ﬁbroin nanoﬁbers (FS) prepared by electrospinning with the objective of improving the hemocompatibility of the ﬁbers for application as scaﬀolds in tissue engineering. The nanoﬁber mats of silk ﬁbroin without (MF-FS) and with (MF- FS/HS) immobilized heparin were characterized through scanning electron microscopy (SEM), Fourier-transform infrared spectroscopy with attenuated total reﬂectance (FTIR-ATR), thermogravimetric analyses (TGA), energy dispersive spectroscopy (EDS), contact angle, chemical analysis, and biological tests. The formation of hydrogen bonds between the silk ﬁbroin and heparin was discussed based on FTIR-ATR spectra. The amount of immobilized heparin was quantiﬁed through papain/N-acetyl-L-cysteine digestion followed by dimethylmethylene blue complexation. Furthermore, the samples with immobilized HS showed higher hydrophilic capability compared to samples without HS due to lower contact angles. It was possible to verify that the capillary end-to-collector distance of 8.5 cm and ﬂow rate of 0.35 mL h -1 used in the electrospinning process at 20 kV are good conditions for obtaining a small average ﬁber diameter maintaining the amount of immobilized heparin on MF-FS/HS in ca. 4% w/w. Biological analysis showed that no hemolysis is provoked by MF-FS and MF-FS/HS mat fragments and those such mats are not toxic to Vero cells. However, the MF-FS/HS showed higher cell growth and proliferation than MF-FS, indicating an improvement in the hemocompatibility of the material due to heparin immobilization. ■ INTRODUCTION The fabrication of nanoﬁbers by electrospinning has received great attention due to their versatility to produce submi- crometer ﬁbers and nanoscale control of the structure, porosity, and orientation. 1 The application of nanoﬁber matrixes as scaﬀolds in tissue engineering have shown great results for building and/or the regeneration of various tissues including bone, cartilage, tendons, blood vessels, and heart valves. Scaﬀolds are three-dimensional (3D) structures that suit the needs of newly growing tissue. 2 Many synthetic and natural polymers have been examined to obtain matrixes for application in tissue engineering. 3 Silk ﬁbroin is a natural biopolymer that is present in the bark of the cocoons produced by the species Bombyx mori. The silk produced by the silkworm has excellent mechanical properties as well as favorable biocompatibility, environmental stability, controlled proteolytic biodegradability, morphologic ﬂexibility, good permeability to water vapor, and minimal inﬂammatory reaction. 4,5 Because of these properties, the silk ﬁbroin is a material of great potential for the generation of biomaterials for diﬀerent uses, including devices for tissue engineering. 5 The biocompatible, biomimetic, mechanical properties close to the tissue are the main consideration for biopolymers use as based material in scaﬀolds for cell culture. In addition, the compatibility of the scaﬀolds with the blood cells is very important for speci ﬁc tissue engineering applications such as artery and veins reconstruction, for instance. Thus, various strategies have been proposed to improve the thrombogenicity of biomaterials, such as the incorporation of ionic groups on the polymer surface, changing the surface properties by grafting techniques and immobiliza- tion of heparin, functionalized dextrans, or biological compounds. 6 In general, immobilization of heparin is the most eﬀective and widely used strategy for improving hemocompatibility through various methods such as coating and mixture grafting. 7 Heparin is a highly anionic glycosami- noglycan isolated by extraction from animal tissues, which are rich in mast cells present in pig intestines. 5 It is clinically used as an anticoagulant to minimize thrombus formation on artiﬁcial organ surfaces. There are two general methods for developing polymeric blood compatible material using heparin. One method uses chemical immobilization of heparin, and the other is focused on the delivery system of heparin. 8 The aim of Received: January 26, 2014 Revised: March 21, 2014 Published: March 26, 2014 Article pubs.acs.org/Biomac © 2014 American Chemical Society 1762 dx.doi.org/10.1021/bm500132g | Biomacromolecules 2014, 15, 1762-1767