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Título/Title/Titulo

BIOCOMPATIBILITY EVALUATION OF PARTICULATE PPy/p-TSA USING ZEBRAFISH AS A MODEL ORGANISM

Introdução/Introduction/Introdución

Zebrafish (Danio rerio) is an animal model increasingly used in biomedical research including human toxicology (Raldúa et al., 2012) and one of the most promising in vivo model systems for toxicity screening (Bohnsack et al., 2012). Recently, zebrafish have been used to evaluate the toxicity of several nanomaterials (Wang et al., 2015) because is a facile model for the rapid evaluation of the potential toxicity and biodistribution of nanomaterials (Fako et al., 2009). After the discovery that electrical signals can regulate cell attachment, proliferation and differentiation (Rivers et al., 2002), researches sought to incorporate conducting polymers into biomaterials to take advantage of electrical stimuli, and the polypyrrole (PPy) has been studied in biomedical applications (Tian et al., 2012).

Objetivos - Metodologia - Resultados - Discussão dos Resultados/Objectives - Methodology - Results - Discussion of Results/Objetivos - Metodología - Resultados - Discusión de los resultados

The present study aimed at analyzing the biocompatibility of PPy doped with p-toluenesulfonic acid ((p-TSA); PPy/p-TSA), after exposition of different concentration of nanomaterial in zebrafish larvae. PPy/p-TSA was synthesized by oxidative polymerization method. The morphology of PPy/p-TSA were measured using Field-Emission Scanning Electron Microscopy (FESEM) and Transmission Electronic Microscopy. The conductivity was determined by four-point probe method and particle size was estimated by FESEM. The Animal Ethics Committee (CEUA 15/00479) approved all the protocols. The zebrafish embryos were exposed to the dispersion PPy/p-TSA from up to 4 hours post-fertilization (hpf) until 144 hpf. Survival Curve: Larvae mortality was evaluated in the groups (Naïve, Vehicle, 25; 100; 250 and 500 µg/mL PPy/p-TSA) in 24, 48, 72, 96, 120 and 144 hpf after particulate PPy/p-TSA exposure (30 larvae per group). Embryo toxicity test: The embryonic spontaneous movement (1 min) were monitored with the aid of a microscope at the time point of 24 hpf (10 embryo per group) and the frequency of the heart bates of embryo/larvae (1 min) were monitored with the aid of a microscope at the time point of 48 hpf (10 embryo/larvae per group). Statistical Analysis: We used Kaplan-Meier method for the survival curve and One-way Analysis of Variance (ANOVA) followed by Tukey's test in the spontaneous movement and cardiac rate. Data were expressed as mean ± S.E. and p<0.05 was considered significant. The morphology of p-TSA-doped PPy showed a granular morphology with irregular spheres particulate. The statistical distribution from FESEM images shows that about 84% of particles ranging 178-518 nm in size, 321 ± 139 nm in average (mean ± S.D., n = 880) and minimum of 23 nm and maximum of 896 nm. The electrical conductivity of PPy/p-TSA is 4.4x 10 -6 S/cm, within the semiconductor range (10 -7 – 10 3 S/cm) (Balint et al., 2014). To evaluate the possible toxicity of the suspensions of PPy/p-TSA in different concentrations to zebrafish, the mortality was analyzed and a significant reduction in the larvae survival in the doses of 500 µg/mL it was noted after 144 h exposure, featuring 70% mortality. The spontaneous movement analysis shows a trend of increasing movements of animals in the groups treated with higher concentrations of the nanomaterial. However, so far has not found any statistical difference in relation to the treated groups when analyzing the average heart beats.

Considerações Finais/Final considerations/Consideraciones finales

Additional experiments are in progress to evaluate the potential inflammatory or toxic of PPy/p-TSA, and will be useful to establish quality in the development of biomaterials in the field of regenerative medicine

Palavras-chave/Key words/Palabras clave

POLYPYRROLE; PPy/p-TSA; BIOCOMPATIBILITY; NANOTOXICITY; ZEBRAFISH