Pharmaceutical Research

, Volume 33, Issue 2, pp 404–416 | Cite as

Liposome Formation Using a Coaxial Turbulent Jet in Co-Flow

  • Antonio P. Costa
  • Xiaoming Xu
  • Mansoor A. Khan
  • Diane J. Burgess
Research Paper



Liposomes are robust drug delivery systems that have been developed into FDA-approved drug products for several pharmaceutical indications. Direct control in producing liposomes of a particular particle size and particle size distribution is extremely important since liposome size may impact cellular uptake and biodistribution.


A device consisting of an injection-port was fabricated to form a coaxial turbulent jet in co-flow that produces liposomes via the ethanol injection method. By altering the injection-port dimensions and flow rates, a fluid flow profile (i.e., flow velocity ratio vs. Reynolds number) was plotted and associated with the polydispersity index of liposomes.


Certain flow conditions produced unilamellar, monodispersed liposomes and the mean particle size was controllable from 25 up to >465 nm. The mean liposome size is highly dependent on the Reynolds number of the mixed ethanol/aqueous phase and independent of the flow velocity ratio.


The significance of this work is that the Reynolds number is predictive of the liposome particle size, independent of the injection-port dimensions. In addition, a new model describing liposome formation is outlined. The significance of the model is that it relates fluid dynamic properties and lipid-molecule physical properties to the final liposome size.


coaxial turbulent jet continuous manufacturing ethanol injection liposome processing monodispersed liposomes unilamellar 



31 phosphorous nuclear magnetic resonance


Cross-sectional area




Cryogenic transmission electron microscopy




Dynamic light scattering




Design of experiment






1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt)




Flow velocity ratio


International conference on harmonisation


Inner diameter


National instruments


Negative stain transmission electron microscopy


Outer diameter


Process analytical technology


Polydispersity index


Combined output flow rate


Reynolds number


Kinematic viscosity



This work was supported by the U.S. FDA (Grant#: HHSF223201310117C). We thank Dr. M. Cantino and Dr. X. Sun from the Biosciences Electron Microscope Laboratory of the Physiology and Neurobiology Department at the University of Connecticut for their work on the negative stain TEM micrographs. The authors would like to acknowledge Dr. Jiwen Zheng and Dr. Yong Wu at the FDA White Oak Nanotechnology Core Facility for instrument use, scientific and technical assistance. A. Costa was an AFPE fellow during the time period of this research.

Conflict of Interest

The authors declare no competing financial interest.


The views expressed are those of authors and do not necessarily represent the official position of the Agency (FDA).

Supplementary material

11095_2015_1798_MOESM1_ESM.docx (16 kb)
ESM 1 (DOCX 15 kb)


  1. 1.
    Batzri S, Korn ED. Single bilayer liposomes prepared without sonication. Biochim Biophys Acta Biomembr. 1973;298(4):1015–9.CrossRefGoogle Scholar
  2. 2.
    ICH Harmonised Tripartite Guideline. Impurities: guideline for residual solvents Q3C(R4), current step 4 version. 2009;1–25.Google Scholar
  3. 3.
    Buchholz S. Future manufacturing approaches in the chemical and pharmaceutical industry. Chem Eng Process. 2010;49:993–5.CrossRefGoogle Scholar
  4. 4.
    FDA. Guidance for industry PAT—a framework for innovative pharmaceutical development, manufacturing, and quality assurance. 2004;1–19.Google Scholar
  5. 5.
    Wagner A, Vorauer-Uhl K, Kreismayr G, Katinger H. The crossflow injection technique: an improvement of the ethanol injection method. J Liposome Res. 2002;12(3):259–70.CrossRefPubMedGoogle Scholar
  6. 6.
    Jahn A, Vreeland WN, Gaitan M, Locascio LE. Controlled vesicle self-assembly in microfluidic channels with hydrodynamic focusing. J Am Chem Soc. 2004;126:2674–5.CrossRefPubMedGoogle Scholar
  7. 7.
    Lim J-M, Swami A, Gilson LM, Chopra S, Choi S, Wu J, et al. Ultra-high throughput synthesis of nanoparticles with homogeneous size distribution using a coaxial turbulent jet mixer. ACS Nano. 2014;8:6056–65.PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Jaafar-Maalej C, Charcosset C, Fessi H. A new method for liposome preparation using a membrane contactor. J Liposome Res. 2011;21:213–20.CrossRefPubMedGoogle Scholar
  9. 9.
    Jahn A, Vreeland WN, DeVoe DL, Locascio LE, Gaitan M. Microfluidic directed formation of liposomes of controlled size. Langmuir. 2007;23:6289–93.CrossRefPubMedGoogle Scholar
  10. 10.
    Hood RR, DeVoe DL, Atencia J, Vreeland WN, Omiatek DM. A facile route to the synthesis of monodisperse nanoscale liposomes using 3D microfluidic hydrodynamic focusing in a concentric capillary array. Lab Chip. 2014;14:2403–9.CrossRefPubMedGoogle Scholar
  11. 11.
    Jahn A, Lucas F, Wepf RA, Dittrich PS. Freezing continuous-flow self-assembly in a microfluidic device: toward imaging of liposome formation. Langmuir. 2013;29:1717–23.CrossRefPubMedGoogle Scholar
  12. 12.
    Greenspan P, Fowler SD. Spectrofluorometric studies of the lipid probe, Nile red. J Lipid Res. 1985;26:781–9.PubMedGoogle Scholar
  13. 13.
    Perrett S, Golding M, Williams WP. A simple method for the preparation of liposomes for pharmaceutical applications: characterization of the liposomes. J Pharm Pharmacol. 1991;43:154–61.CrossRefPubMedGoogle Scholar
  14. 14.
    Khattab IS, Bandarkar F, Fakhree MAA, Jouyban A. Density, viscosity, and surface tension of water+ethanol mixtures from 293 to 323K. Korean J Chem Eng. 2012;29:812–7.CrossRefGoogle Scholar
  15. 15.
    Kwon S, Seo I. Reynolds number effects on the behavior of a non-buoyant round jet. Exp Fluids. 2005;38:801–12.CrossRefGoogle Scholar
  16. 16.
    Or CM, Lam KM, Liu P. Potential core lengths of round jets in stagnant and moving environments. J Hydro Environ Res. 2011;5:81–91.CrossRefGoogle Scholar
  17. 17.
    Chu P, Lee J, Chu V. Spreading of turbulent round jet in Coflow. J Hydraul Eng. 1999;125:193–204.CrossRefGoogle Scholar
  18. 18.
    Zhao E, Benson GC, Lu BCY. Excess enthalpies of ternary mixtures of ethanol+water+sodium chloride or zinc chloride at 298.15 K. J Chem Eng Data. 1995;40:665–8.CrossRefGoogle Scholar
  19. 19.
    Lasic DD. The mechanism of vesicle formation. Biochem J. 1988;256:1–11.PubMedCentralCrossRefPubMedGoogle Scholar
  20. 20.
    Patra M, Salonen E, Terama E, Vattulainen I, Faller R, Lee BW, et al. Under the influence of alcohol: the effect of ethanol and methanol on lipid bilayers. Biophys J. 2006;90:1121–35.PubMedCentralCrossRefPubMedGoogle Scholar
  21. 21.
    Touitou E, Dayan N, Bergelson L, Godin B, Eliaz M. Ethosomes—novel vesicular carriers for enhanced delivery: characterization and skin penetration properties. J Control Release. 2000;65:403–18.CrossRefPubMedGoogle Scholar
  22. 22.
    Szoka F, Papahadjopoulos D. Comparative properties and methods of preparation of lipid vesicles (Liposomes). Annu Rev Biophys Bioeng. 1980;9:467–508.CrossRefPubMedGoogle Scholar
  23. 23.
    Marsh D. Intrinsic curvature in normal and inverted lipid structures and in membranes. Biophys J. 1996;70(5):2248–55.PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Pons M, Foradada M, Estelrich J. Liposomes obtained by the ethanol injection method. Int J Pharm. 1993;95:51–6.CrossRefGoogle Scholar
  25. 25.
    Jaafar-Maalej C, Diab R, Andrieu V, Elaissari A, Fessi H. Ethanol injection method for hydrophilic and lipophilic drug-loaded liposome preparation. J Liposome Res. 2010;20:228–43.CrossRefPubMedGoogle Scholar
  26. 26.
    Schubert MA, Müller-Goymann CC. Solvent injection as a new approach for manufacturing lipid nanoparticles—evaluation of the method and process parameters. Eur J Pharm Biopharm. 2003;55:125–31.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Antonio P. Costa
    • 1
  • Xiaoming Xu
    • 2
  • Mansoor A. Khan
    • 2
  • Diane J. Burgess
    • 1
  1. 1.Department of Pharmaceutical SciencesUniversity of ConnecticutStorrsUSA
  2. 2.FDA/CDER/DPQRSilver SpringUSA

Personalised recommendations