Advertisement

Pharmaceutical Research

, 35:229 | Cite as

Sustained Release from Ionic-Gradient Liposomes Significantly Decreases ETIDOCAINE Cytotoxicity

  • Juliana Damasceno Oliveira
  • Lígia Nunes de Morais Ribeiro
  • Gustavo Henrique Rodrigues da Silva
  • Bruna Renata Casadei
  • Verônica Muniz Couto
  • Elizabeth Ferreira Martinez
  • Eneida de Paula
Research Paper
  • 136 Downloads

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 (NH4)2SO4 - inside gradient - were prepared for the encapsulation of 0.5% EDC. Dynamic light scattering, nanotracking analysis, transmission electron microscopy and electron paramagnetic resonance were used to characterize: nanoparticles size, polydispersity, zeta potential, concentration, morphology and membrane fluidity. Release kinetics and in vitro cytotoxicity tests were also performed.

Results

IGLEDC showed average diameters of 172.3 ± 2.6 nm, low PDI (0.12 ± 0.01), mean particle concentration of 6.3 ± 0.5 × 1012/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, induced 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. IGLEDC 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

IC50

Half maximal inhibitory concentration of cell viability

IGL

Ionic gradient liposomes

IGLEDC

Etidocaine-containing sulphate gradient liposomes

LA

Local anesthetic

LUV

Large unilamellar vesicle

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

NTA

Nanotracking analysis

TEM

Transmission electron microscopy

References

  1. 1.
    Wang G-K, Strichartz GR. State-dependent inhibition of sodium channels by local anesthetics: a 40-year evolution. Biochem (Mosc) Suppl Ser A Membr Cell Biol. 2012;6(2):120–7.CrossRefGoogle Scholar
  2. 2.
    Lirk P, Picardi S, Hollmann MW. Local anaesthetics: 10 essentials. Eur J Anaesthesiol. 2014;31(11):575–85.CrossRefGoogle Scholar
  3. 3.
    Strichartz GR, Sanchez V, Arthur GR, Chafetz R, Martin D. Fundamental properties of local anesthetics. II. Measured octanol: buffer partition coefficients and pKa values of clinically used drugs. Anesth Analg. 1990;71(2):158–70.CrossRefGoogle Scholar
  4. 4.
    Covino BG, Vassalo HG. Local anesthetics: mechanisms of action and clinical use. Rio de Janeiro: Colina Editora; 1985.Google Scholar
  5. 5.
    Garciat DG. Etidocaine - a long-acting anesthestic agent: review of the literature. Anesth Prog. 1972;29(1):12–3.Google Scholar
  6. 6.
    Lund P, Cewik J, Gannon R. Etidocaine (Duranest®): a clinical and laboratory evaluation. Acta Anaesthesiol Scand Suppl. 1974;18(3):176–88.CrossRefGoogle Scholar
  7. 7.
    Discontinued Drug Product List. US food and drug administration. Docket n° 2008-P-0527. September 25th, 2008. Available from: https://www.accessdata.fda.gov/scripts/cder/ob/results_product.cfm?Appl_Type=N&Appl_No=017751. Acessed March 02, 2018.
  8. 8.
    Determination that Duranest® (etidocaine hydrochloride) injection, 0.5%, and five other Duranest® drug products were not withdrawn from sale for reasons of safety or effectiveness. FR. Federal Register / Vol. 77, No. 49 / March 13th, 2012 / Notices T. Vol. 77. 2012.Google Scholar
  9. 9.
    Allen T, Cullins P. Liposomal drug delivery systems: from concept to clinical applications. Adv Drug Deliv Rev. 2013;65(1):36–48.CrossRefGoogle Scholar
  10. 10.
    Santamaria CM, Woodruff A, Yang R, Kohane DS. Drug delivery systems for prolonged duration local anesthesia. Mater Today. 2017;20(1):22–31.CrossRefGoogle Scholar
  11. 11.
    Grant GJ, Barenholz Y, Piskoun B, Bansinath M, Turndorf H, Bolotin EM. DRV liposomal bupivacaine: preparation, characterization, and in vivo evaluation in mice. Pharm Res. 2001;18(3):336–43.CrossRefGoogle Scholar
  12. 12.
    de Paula E, Cereda CMS, Fraceto LF, de Araújo DR, Franz-Montan M, Tofoli GR, et al. Micro and nanosystems for delivering local anesthetics. Expert Opin Drug Deliv. 2012;9(12):1505–24.Google Scholar
  13. 13.
    Mura P, Capasso G, Maestrelli F, Furlanetto S. Optimization of formulation variables of benzocaine liposomes using experimental design. J Liposome Res. 2008;18(2):113–25.CrossRefGoogle Scholar
  14. 14.
    Akbarzadeh A, Rezaei-sadabady R, Davaran S, Joo SW, Zarghami N. Liposome : classification, preparation, and applications. Nanoescale Res Lett. 2013;8(1):102.CrossRefGoogle Scholar
  15. 15.
    da Silva CMG, Franz-Montan M, Limia CEG, Ribeiro LN de M, Braga MA, Guilherme VA, et al. Encapsulation of ropivacaine in a combined (donor-acceptor, ionic-gradient) liposomal system promotes extended anesthesia time. PLoS One. 2017;12(10):1–16.Google Scholar
  16. 16.
    Barenholz Y, Haran G. Method of amphiphatic drug loading in liposome by amonium ion gradient. US Patent. 1994;5:316–771.Google Scholar
  17. 17.
    Bolotin EM, Cohen R, Bar LK, Emanuel N, Ninio S, Barenholz Y, et al. Ammonium sulfate gradients for efficient and stable remote loading of amphipathic weak bases into liposomes and ligandoliposomes. J Liposome Res. 1994;4(1):455–79.CrossRefGoogle Scholar
  18. 18.
    Grant GJ, Barenholz Y, Bolotin EM, Bansinath M, Turndorf H, Piskoun B, et al. A novel liposomal bupivacaine formulation to produce ultralong-acting analgesia. Anesthesiology. 2004;101(1):133–7.CrossRefGoogle Scholar
  19. 19.
    Clerc S, Barenholz Y. Loading of amphipathic weak acids into liposomes in response to transmembrane calcium acetate gradients. BBA - Biomembr. 1995;1240(2):257–65.CrossRefGoogle Scholar
  20. 20.
    da Silva CMG, Fraceto LF, Franz-Montan M, Couto VM, Casadei BR, Cereda CMS, et al. Development of egg PC/cholesterol/α-tocopherol liposomes with ionic gradients to deliver ropivacaine. J Liposome Res. 2016;26(1):1–10.CrossRefGoogle Scholar
  21. 21.
    de Paula E, Schreier S. Use of a novel method for determination of partition coefficients to compare the effect of local anesthetics on membrane structure. Biochim Biophys Acta. 1995;1240(1):25–33.CrossRefGoogle Scholar
  22. 22.
    ICH. Validation of analytical procedures: text and methodology Q2(R1). Geneva: Intl Conf. Harmon; 2005.Google Scholar
  23. 23.
    Tu S, Mcginnis T, Krugner-Higby L, Heath TD. A mathematical relationship for hydromorphone loading into liposomes with trans-membrane ammonium sulfate gradients. J Pharm Sci. 2010;99(6):2672–80.CrossRefGoogle Scholar
  24. 24.
    Chen PS, Toribara TY, Warner H, Chen PS Jr, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chem. 1956;28(11):1756–8.CrossRefGoogle Scholar
  25. 25.
    Allain CC, Poon LS, Chan CSG, Richmond W, Fu PC. Enzymatic determination of total serum cholesterol. Clin Chem. 1974;20(4):470–5.Google Scholar
  26. 26.
    Zucker D, Marcus D, Barenholz Y, Goldblum A. Liposome drug’s loading efficiency: a working model based on loading conditions and drug’s physicochemical properties. J Control Release. 2009;139(1):73–80.CrossRefGoogle Scholar
  27. 27.
    Hubbell WL, McConnell HM. Molecular motion in spin-labeled phospholipids and membranes. J Am Chem Soc. 1971;93(2):314–26.CrossRefGoogle Scholar
  28. 28.
    Schreier S, Polnaszek CF, Smith I. Spin labels in membranes problems in practice. Biochim Biophys Acta. 1978;515(4):395–436.CrossRefGoogle Scholar
  29. 29.
    Franz TJ. Percutaneous absorption. On the relevance of in vitro data. J Invest Dermatol. 1975;64(3):190–5.CrossRefGoogle Scholar
  30. 30.
    Mendyk A, Jachowicz R. Unified methodology of neural analysis in decision support systems built for pharmaceutical technology. Expert Syst Appl. 2007;32(4):1124–31.CrossRefGoogle Scholar
  31. 31.
    Ribeiro LNM, Franz-Montan M, Breitkreitz MC, Alcântara ACS, Castro SR, Guilherme VA, et al. Nanostructured lipid carriers as robust systems for topical lidocaine-prilocaine release in dentistry. Eur J Pharm Sci. 2016;93:192–202.CrossRefGoogle Scholar
  32. 32.
    Martinez EF, Araújo VC. In vitro immunoexpression of extracellular matrix proteins in dental pulpal and gingival human fibroblasts. Int Endod J. 2004;37(11):749–55.CrossRefGoogle Scholar
  33. 33.
    Gasco MR. Lipid nanoparticles: perspectives and challenges. Adv Drug Deliv Rev. 2007;59(6):377–8.CrossRefGoogle Scholar
  34. 34.
    Mohanraj V, Chen Y, Chen M. Nanoparticles – a review. Trop J Pharm Res Trop J Pharm Res. 2006;5(1):561–73.Google Scholar
  35. 35.
    Bhattacharjee S. DLS and zeta potential – what they are and what they are not? J Control Release. 2016;235:337–51.CrossRefGoogle Scholar
  36. 36.
    Fraceto LF, Spisni A, Schreier S, de Paula E. Differential effects of uncharged aminoamide local anesthetics on phospholipid bilayers, as monitored by 1H-NMR measurements. Biophys Chem. 2005;115(1):11–8.Google Scholar
  37. 37.
    Ribeiro LNM, Couto VM, Fraceto LF, De Paula E. Use of nanoparticle concentration as a tool to understand the structural properties of colloids. Sci Rep. 2018;8(1):1–8.CrossRefGoogle Scholar
  38. 38.
    Haran G, Cohen R, Bar LK, Barenholz Y. Transmembrane ammonium sulfate gradients in liposomes produce efficient and stable entrapment of amphipathic weak bases. BBA - Biomembr. 1993;1151(2):201–15.CrossRefGoogle Scholar
  39. 39.
    de Paula E, Schreier S, Jarrell HC, Fraceto LF. Preferential location of lidocaine and etidocaine in lecithin bilayers as determined by EPR, fluorescence and 2H NMR. Biophys Chem. 2008;132(1):47–54.CrossRefGoogle Scholar
  40. 40.
    Gubernator J. Active methods of drug loading into liposomes: recent strategies for stable drug entrapment and increased in vivo activity. Expert Opin Drug Deliv. 2011;8(5):565–80.CrossRefGoogle Scholar
  41. 41.
    Yeagle PL. The structure of biological membranes. 3rd ed. Boca Raton: CRC Rress; 2012.Google Scholar
  42. 42.
    Li J, Wang X, Zhang T, Wang C, Huang Z, Luo X, et al. A review on phospholipids and their main applications in drug delivery systems. Asian J Pharm Sci. 2014;10:81–98.CrossRefGoogle Scholar
  43. 43.
    Zhong H, Deng Y, Wang X, Yang B. Multivesicular liposome formulation for the sustained delivery of breviscapine. Int J Pharm. 2005;301(1–2):15–24.CrossRefGoogle Scholar
  44. 44.
    Grant SA. The holy grail: long-acting local anaesthetics and liposomes. Best Pract Res Clin Anaesthesiol. 2002;16(2):345–52.CrossRefGoogle Scholar
  45. 45.
    Couto VM, Prieto MJ, Igartúa DE, Feas DA, Ribeiro LN, Silva CM, et al. Dibucaine in ionic-gradient liposomes: biophysical, toxicological and activity characterisation. J Pharm Sci. 2018;107:2411–9.CrossRefGoogle Scholar
  46. 46.
    Costa P, Lobo JM. Modeling and comparison of dissolution profiles. Eur J Pharm Sci. 2001;13(2):123–33.CrossRefGoogle Scholar
  47. 47.
    Malheiros SVP, Pinto LMA, Gottardo L, Yokaichiya DK, Fraceto LF, Meirelles NC, et al. A new look at the hemolytic effect of local anesthetics, considering their real membrane/water partitioning at pH 7.4. Biophys Chem. 2004;110(3):213–21.CrossRefGoogle Scholar
  48. 48.
    Adade AB, Chignell D, Vanderkooi G. Local anesthetics: a new class of partial inhibitors of mitochondrial ATPase. J Bioenerg Biomembr. 1984;16(5–6):353–63.CrossRefGoogle Scholar
  49. 49.
    Chang Y-C, Liu C-L, Chen M-J, Hsu Y-W, Chen S-N, Lin C-H, et al. Local anesthetics induce apoptosis in human breast tumor cells. Anesth Analg. 2014;118(1):116–24.CrossRefGoogle Scholar
  50. 50.
    Torchilin VP. Interview with Vladimir P Torchilin: liposomal carriers for drug delivery. Ther Deliv. 2013;4(5):537–8.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • 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
  1. 1.Department of Biochemistry and Tissue Biology, Institute of BiologyUniversity of Campinas – UnicampCampinasBrazil
  2. 2.Department of BiophysicsFederal University of São Paulo – UNIFESP, São PauloSão PauloBrazil
  3. 3.Department of Oral PathologySão Leopoldo Mandic Institute and Research CenterSão PauloBrazil

Personalised recommendations