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Liposomes pp 369-383 | Cite as

Viscometric Analysis of DNA-Lipid Complexes

  • Sadao HirotaEmail author
  • Nejat Düzgüneş
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 606)

Abstract

DNA-cationic lipid complexes, “lipoplexes”, are used as gene carriers for molecular biology and gene therapy applications. Colloidal properties of lipoplexes can be determined by viscometric analysis. (1) The shape parameter of lipoplexes can be one of the factors affecting transfection efficiency; (2) the volume fraction of free liposomes remaining after lipoplex formation can be used as an index of purity of the lipoplex product; (3) the shear dependence of the viscosity of a diluted lipoplex suspension can be used as a macroscopic shape factor: (4) the attraction force parameter between particles can be a colloidal stability factor. These properties should be characterized and specified for process control of lipoplex production and quality control of lipoplex products.

We describe an automated mini-capillary viscometer for a sample volume of 0.5 ml, and its application to the characterizations of lipoplexes. We show a procedure for viscosity measurements and provide a calculation using complexes of plant DNA-distearyldimethylammonium chloride (DDAC) at a charged ratio of 1:4 (−/+), in which the amount of DNA is less than 250 µg. The prolate/ellipsoidal axial ratio, a/b, was found to be 70. Determination of the shape parameter with a/b is found to be better than that with other shape parameters, e.g., α of the Sakurada equation, because fractionation of the particle size is not necessary. By the proposed method, colloidal parameters of lipoplexes and bioactive polymer complexes are characterized quantitatively.

Key words

Shape parameter Ellipsoid Liposomes Lipoplexes Viscosity Capillary viscometer Quality control in large scale production 

Symbols

a

Longer semi-diameter of ellipsoid (cm)

b

Shorter semi-diameter of ellipsoid (cm)

c

Concentration in molality (mol/kg)

L

Length of capillary (cm)

k′

Huggins coefficient

k

Attracting force parameter between particles

k0

kat zero shear

P

Pressure difference between both ends of capillary (dyn/cm2)

r

Distance from axis of capillary (cm)

R

Radius of capillary (cm)

Re

Reynolds number

S

Shear stress (dyn/cm2)

SR

Shear stress at inner wall of capillary (dyn/cm2)

S0

Yield stress (dyn/cm2)

t

Time (s)

u

Linear velocity (cm/s) at distance r from axis of capillary (cm/s)

U

Volume velocity, V/t, (ml/s)

α

Shape parameter of the Sakurada equation

φ

Volume fraction, volume of particles / volume of suspension

φav

Average volume fraction

φi

Volume fraction of inner aqueous phase

φnet

Net volume fraction without inner aqueous phase

φc

Volume fraction of cationic liposomes

μred

Reduced viscosity, μred = η sp/c (L/g or L/mol)

[μ]

Intrinsic viscosity, reduced viscosity at infinite dilution (L/g or L/mol)

η

Viscosity of sample liquid (poise)

η0

Viscosity of solvent or suspending medium (poise)

ηrel

Relative viscosity, η rel =η/η o

ηsp

Specific viscosity, η sp =(η rel-1)

ηred

Non-dimensional reduced viscosity, η red = η sp/φ

[η]

Non-dimensional intrinsic viscosity, [η] = lim(φ→0)η red

Notes

Acknowledgments

We wish to thank Dr. Y. Sun at the Harbin Institute of Technology (China), for preparation of the lipoplexes and the flow time measurements of viscosity, and Mr. Y. Takaoka at Tokyo Denki University for automation of the mini-capillary viscometer.

References

  1. 1.
    Sun Y, Li X, Düzgünes¸ N, Takaoka Y, Ohi S, Hirota S (2003) The shape parameter of liposomes and DNA-lipid complexes determined by viscometry utilizing small sample volumes. Biophys J. 85(2):1223-1232CrossRefPubMedGoogle Scholar
  2. 2.
    Oberle V, Bakowsky U, Zuhorn IS, Hoekstra D (2000) Lipoplex formation under equilibrium conditions reveals a three-step mechanism. Biophys J 79(3):1447-1454CrossRefPubMedGoogle Scholar
  3. 3.
    Felgner PL, Gadek TR, Holm M, Roman R, Chan HW, Wenz M, Northrop JP, Ringold GM, Danielsen M (1987) Lipofection: a highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA 84(21):7413-7417CrossRefPubMedGoogle Scholar
  4. 4.
    Sternberg B. (1998) Ultrastructural morphology of cationic liposome-DNA complexes for gene therapy. In: Medical Applications of Liposomes. Lasic DD, Papahadjopoulos D (Eds), 395. Elsevier, AmsterdamGoogle Scholar
  5. 5.
    Einstein A (1906) Ann Phys 1928:9Google Scholar
  6. 6.
    Simha R (1940) Viscosity and the shape of protein molecules. Science 92:132-133CrossRefPubMedGoogle Scholar
  7. 7.
    Hirota S (2004) Viscometric determi­nation of axial ratio of ellipsoidal DNA-lipid ­complex. Methods Enzymol. 375: 177-199Google Scholar
  8. 8.
    Tanford C (1961) Physical Chemistry of Macro­molecules, Chapter 6 Transport properties, viscosity. John Wiley & Sons, New York 331-393Google Scholar
  9. 9.
    Huggins ML (1942) The viscosity of dilute solutions of long-chain molecules. IV. Dependence on concentration. J Am Chem Soc 64:2716-2718CrossRefGoogle Scholar
  10. 10.
    Hirota S, de Ilarduya CT, Barron LG, Szoka FC Jr (1999) Simple mixing device to reproducibly prepare cationic lipid-DNA complexes (lipoplexes). Biotechniques 27(2):286-90PubMedGoogle Scholar
  11. 11.
    Sakurada I (1924) Kogyokagakuzasshi 38:383-398 (in Japanese)Google Scholar
  12. 12.
    Lasic D, Strey H, Stuart MCA, Podgornik R, Frederik PM (1997) The structure of DNA-liposome complexes. J Am Chem Soc 119(4):832-833CrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Material Science, School of EngineeringTokyo Denki UniversityTokyoJapan
  2. 2.Department of Microbiology, Arthur A. Dugoni School of DentistryUniversity of the PacificSan FranciscoUSA

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