FULL PAPER Macromolecular Symposia Polycaprolactone www.ms-journal.de Biocompatible PCL/PLGA/Polypyrrole Composites for Regenerating Nerves Cristina Lorenski Ferreira, Cristhiane Alvim Valente, Mara Lise Zanini, Bruna Sgarioni, Pedro Henrique Ferreira Tondo, Pedro Cesar Chagastelles, Jefferson Braga, Maria Martha Campos, Jose Antonio Malmonge, and Nara Regina de Souza Basso* 1. Introduction In the field of regenerative medicine, many studies have focused on regenerating peripheral nerves because clinical problems and functional loss of organs are In the field of tissue engineering, studies are actively developing polymeric biomaterials affecting an increasing number of people. Biocompatible polymers can be – for regenerating peripheral nerves.[1 3] potentially used for producing biocompatible tubes in order to aid the Several biocompatible tubular products regeneration of peripheral nerves. This study aims to prepare polymeric approved by the US Food and Drug composites based on polycaprolactone (PCL), poly (lactic-co-glycolic acid) (PLGA), Administration (FDA) are commercially and polypyrrole fibers (PPy) capable of acting as a conduit for regenerating available for repairing nerve tissues, includ- 1 peripheral nerves. Polypyrrole is synthesized by oxidative chemical polymerization ing NeuroTube made of polyglycolic acid TM with p-toluenesulfonic acid monohydrate (PTSA) as a doping agent. PCL/PLGA and Neurolac made of poly (D, L-lactic acid-co-e-caprolactone).[4] blends (100:0, 90:10, 80:20, and 70:30) (m/m) and PCL/PLGA composites with Biodegradable polyesters such as poly 10% PPy fibers were prepared by the solvent casting method. PPy with a (lactic-co-glycolic acid) (PLGA), polycapro- À À diameter of 88–974 nm showed electrical conductivity of 2.0 Â 10 1 Scm 1.The lactone (PCL), and their blends have been nontoxic composite films with hydrophilic and porous surfaces presented a widely used for preparing biocompatible [2,4–7] thermal stability and degradation period that were suitable for potential use in tubes. PLGA is widely used as a substrate for nerve regeneration owing to the regeneration of peripheral nerves. its biodegradability, nontoxicity, and film- forming ability.[4] PLGA-based materials show micropores on the surface that C. L. Ferreira, C. A. Valente, N. R. de Souza Basso increase nutrient permeability, thus favoring cell adhesion S~ Programa de Pos-graduacao em Engenharia e Tecnologia de Materiais, and proliferation.[8,9] PCL, too, is suitable for manufacturing Pontifícia Universidade Catolica do Rio Grande do Sul Porto Alegre, RS, Brazil such tubes because it has low toxicity, good mechanical E-mail: [email protected] properties, slow degradation (>1 year), and excellent compati- M. L. Zanini, N. R. de Souza Basso bility for preparing blends with other biocompatible poly- Escola de Ci^encias mers.[3,4,10] Polymers are combined to obtain biocompatible Pontifícia Universidade Catolica do Rio Grande do Sul tubes that show properties not attainable by any of the Porto Alegre, RS, Brazil constituents alone.[11] PCL/PLGA blends with different ratios B. Sgarioni, P. H. Ferreira Tondo have been considered promising for medical applications owing Escola Politecnica to their biocompatibility, slow degradability, and surface Pontifícia Universidade Catolica do Rio Grande do Sul [1,12,13] Porto Alegre, RS, Brazil topography that favors cell adhesion and proliferation. P. C. Chagastelles, M. M. Campos Conductivepolymersarealsoconsideredattractivematerialsfor Instituto de Toxicologia e Farmacologia medical applications because various biological tissues respond to Pontifícia Universidade Catolica do Rio Grande do Sul electric fields and stimuli. Studies have shown that conductive Porto Alegre, RS, Brazil polymers have good ability to support and modulate the growth of J. Braga several types of cells such as nerve cells and bone cells.[6,14] S~ ^ Programa de Pos-graduacao em Medicina e Ciencias da Saude Polypyrrole (PPy) is the most studied conductive polymer in Pontifícia Universidade Catolica do Rio Grande do Sul Porto Alegre, RS, Brazil vitro and in vivo owing to its biocompatibility and easy synthesis [4,6] J. A. Malmonge route. However, polypyrrole has poor mechanical properties Universidade Estadual Paulista (UNESP) and is not biodegradable. On the other hand, biodegradable Faculdade de Engenharia PCL/PLGA blends have suitable mechanical properties (flexibil- Campus de Ilha Solteira ity) for preparing biocompatible tubes, but they are insulating SP, Brazil materials. Thus, the addition of a small amount of the PPy DOI: 10.1002/masy.201800028 conducting polymer to the PCL/PLGA blends results in a Macromol. Symp. 2019, 383, 18000281800028 (1 of 8) © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim Macromolecular Symposia www.advancedsciencenews.com www.ms-journal.de polymeric material with mechanical and electrical properties USC-2500A model) during 4 h at room temperature. After, the appropriate to support and stimulate the growth of nerve cells. polymers dispersion were poured into glass Petri-dishes (5.5 cm PLGA fibers prepared by electrospinning and coated with PPy in diameter). The solvent was evaporated at room temperature showed better electrical activity and cellular interactions for 48 h and then the films were vacuum-dried for 8 h. The films compared to fibers without PPy.[13] Similarly, a PCL fiber coated of the PCL/PLGA/PPy composites were prepared by dispersion with conducting layers was prepared by adding pyrrole to the of PPy fibers to the PCL/PLGA blends according to the polymer solution for electrospinning and then polymerizing in methodology described previously. These polymeric systems fi FeCl3 solution containing an anionic surfactant and added were maintained for 8 h in ultrasonic bath due to the dif culty of pyrrole monomer.[15] Conducting tubes with a PCL-PPy inner dispersion of the filler. In the preparation of the PCL/PPy layer and PLGA outer layer were prepared, and the results film, the methodology used was the same. For all composites showed that when electrically stimulated, these tubes increased films, the amount of PPy added was 10% (m/m) relative to the the axon length growth rate by 21% after 3 days.[16] mass of the biodegradable polymer. In addition to biocompatibility, it is desirable that the material should show topographic characteristics such as high poros- ity.[4,17] The surface must be hydrophilic, because implants with 2.2. In Vitro Degradation hydrophobic surfaces may be rejected owing to the adhesion of monocytes to these surfaces. Furthermore, the material should The degradation experiments were carried out based on ASTM show an optimum degradation rate that must be slow enough to F1635-11 (2011).[21] The film samples cutted into 5-mm diameter provide a support for cell growth but fast enough not to disturb disks was dipped in tubes containing 5 mL of PBS solution, and the regeneration process.[4] incubated at 37 C temperature and 60 rpm-stirring. Each batch In the present study, PPy with fiber morphology was contained six specimens for each evaluated incubation time. The dispersed in PCL/PLGA blends by a solvent casting method specimens were removed from PBS solution after 30, 60, 90, 120, for preparing multifunctional tubes for regenerating peripheral and 150 days. They were carefully washed with deionized water, nerves. The polymeric biomaterials were then examined using vacuum-dried to a constant weight, and then weighed to scanning electron microscopy, water contact angle, thermogra- determine mass loss (in mg). The percentages of mass loss were vimetric analysis (TG), in vitro degradation, cell viability, and calculated from the following Equation (1): electrical conductivity measurements by the two-probe and four- probe methods. The biocompatible films based on PCL and Mass LossðÞ¼% 100 Â ðÞM0 À Mt =M0 ð1Þ PLGA with PPy fibers show appropriate characteristics for tissue M M engineering applications. where 0 is the mass before degradation and t is the dry mass after each time of degradation. Additionally, the pH of the removed PBS was measured after each time of degradation, in a 2. Experimental Section pH-meter (Digimed, DM-20 model). The presented values represent the average of six specimens Æ standard error. Poly(L-lactic-co-glycolic acid) (PLGA; Purasorb PLG8523 (85/15) À L-lactide/glycolide copolymer; inherent viscosity ¼ 2.38 dL g 1 in chloroform) was supplied by Corbion Purac (Gorinchem, the 2.3. Characterization Techniques of Polypyrrole and PCL/PLGA À Netherlands). PCL (molecular weight [Mn] ¼ 80 000 g mol 1), and PCL/PLGA/PPy Films p pyrrole monomer (Py; 98%), ferric chloride (FeCl3; 97%), - toluenesulfonic acid monohydrate (PTSA; 98%), phosphate The morphology of PPy powder and the films produced were buffered saline (PBS; pH ¼ 7.4), and 3-(4,5-dimethylthiazol-2-yl)- analyzed using Field-Emission Scanning Electron Microscopy 2,5-diphenyltetrazolium bromide (MTT) were supplied by (FESEM; FEI-Inspect F50 instrument). All samples were coated – Sigma Aldrich company. Chloroform (CHCl3) was supplied with a thin layer of gold. The fibers diameter was estimated from by Synth (Diadema, SP, Brazil). The reagents were used as FESEM and analyzed using NIH ImageJ 1.36b software for received, except pyrrole, which was distilled for subsequent treatment and measurement randomly of the fibers. In order to polymerization. represent the distribuition of PPy fibers diameters 126 measures were collected from three images of FESEM. The PPy electrical conductivity was determined by the