RESEARCH PAPER Sustained Release from Ionic-Gradient Liposomes Significantly Decreases ETIDOCAINE Cytotoxicity Juliana Damasceno Oliveira 1 & Lígia Nunes de Morais Ribeiro 1 & Gustavo Henrique Rodrigues da Silva 1 & Bruna Renata Casadei 2 & Verônica Muniz Couto 1 & Elizabeth Ferreira Martinez 3 & Eneida de Paula 1 Received: 12 June 2018 /Accepted: 27 September 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 ABSTRACT Purpose Etidocaine (EDC) is a long lasting local anesthetic, which alleged toxicity has restricted its clinical use. Liposomes can prolong the analgesia time and reduce the toxicity of local anesthetics. Ionic gradient liposomes (IGL) have been proposed to increase the upload and prolong the drug release, from liposomes. Methods First, a HPLC method for EDC quantification was validated. Then, large unilamellar vesicles composed of hydrogenated soy phosphatidylcholine:cholesterol with 250 mM (NH 4 ) 2 SO 4 - inside gradient - were prepared for the encapsulation of 0.5% EDC. Dynamic light scattering, nano- tracking analysis, transmission electron microscopy and electron paramagnetic resonance were used to characterize: nanopar- ticles size, polydispersity, zeta potential, concentration, mor- phology and membrane fluidity. Release kinetics and in vitro cytotoxicity tests were also performed. Results IGL EDC showed average diameters of 172.3 ± 2.6 nm, low PDI (0.12 ± 0.01), mean particle concentration of 6.3 ± 0.5 × 10 12 /mL and negative zeta values (-10.2 ± 0.4 mV); parameters that remain stable during storage at 4°C. The formulation, with 40% encapsulation efficiency, in- duced the sustained release of EDC (ca. 24 h), while reducing its toxicity to human fibroblasts. Conclusion A novel formulation is proposed for etidocaine that promotes sustained release and reduces its cytotoxicity. IGL EDC can come to be a tool to reintroduce etidocaine in clinical use. KEY WORDS drug-delivery . etidocaine . ionic gradient liposomes . local anesthesia ABBREVIATIONS Cho Cholesterol DDS Drug delivery system DLS Dynamic light scattering EDC Etidocaine EPR Electron paramagnetic resonance HSPC Hydrogenated soy phosphatidylcholine IC 50 Half maximal inhibitory concentration of cell viability IGL Ionic gradient liposomes IGL EDC Etidocaine-containing sulphate gradient liposomes LA Local anesthetic LUV Large unilamellar vesicle MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo- lium bromide NTA Nanotracking analysis TEM Transmission electron microscopy INTRODUCTION Local anesthetics (LA) prevent neural conduction in a revers- ible way, by impeding the influx of sodium ions trough the voltage-gated channels of excitable membranes (1,2). N-(2,6-dimethylphenyl)-2-[ethyl(propyl)amino]butana- mide, etidocaine (EDC) is an aminoamide LA, with a pKa = 7.8 (3). Its structure is similar to that of lidocaine, only differing by the propyl substituent linked to the amine group and the ethyl group on the α-carbon of the intermediate chain (4). Such hydrocarbon groups led to an increase in lipophilicity (regarding lidocaine), higher plasma protein bound fraction and longer time of action (4,5). * Eneida de Paula depaula@unicamp.br 1 Department of Biochemistry and Tissue Biology, Institute of Biology, University of Campinas – Unicamp, P .O. Box – 6109, CEP , Campinas, SP 13083-862, Brazil 2 Department of Biophysics, Federal University of São Paulo – UNIFESP , São Paulo, São Paulo, Brazil 3 Department of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas, São Paulo, Brazil Pharm Res (2018) 35:229 https://doi.org/10.1007/s11095-018-2512-4