Topics Covered
Background
Gene Therapy
Liposomes in Gene
Therapy
Cationic Liposomes
Zeta Potential and Photon Correlation Spectroscopy
(PCS) in Optimisation of Plasmid:Liposome
Complexes
Applications
Optimisation of the Compacting
Agent
Determination of the Charge of Liposome/DNA
Complexes
Development of More Efficient Formulations
Background
Gene Therapy
Gene therapy is the process by which genetic material is delivered, by means
of a vector, to patients for a therapeutic purpose. Vectors are delivery
vehicles - usually a virus or a liposome - used to transport the genetic
material to target cells in the body.
Liposomes in Gene Therapy
Both cationic and anionic liposomes are currently being investigated as
vectors for gene therapy and their effectiveness in transfection is being
studied by research groups and pharmaceutical companies.
Cationic Liposomes
Cationic liposomes (positively charged) are complexed with DNA (plasmids)
Fig. 1.The liposome:DNA ratio is seen to be essential for optimal
transfection.
Figure 1. Cationic liposomes (positively charged) are
complexed with DNA (plasmids)
Zeta Potential and Photon Correlation
Spectroscopy (PCS) in Optimisation of Plasmid:Liposome Complexes
Zeta potential measurements can be used to optimise the ratio
required for particular liposomes with various plasmids Fig 2.The plot also
shows the z-average diameters of the complex formed at various plasmid:liposome
ratios obtained from photon correlation spectroscopy (PCS) measurements.When the
complex has either a high negative or positive zeta potential, the size is
around 90nm. However, when the zeta
potential approaches the iso-electric point, the z-average diameter
increases indicating aggregation of the complex. Zeta
potential measurements in conjunction with PCS sizing measurements allows
for detailed characterisation of such plasmid: liposome complexes.
Figure 2. Zeta potential measurements can be used to
optimise the ratio required for particular liposomes with various plasmids.
Applications
- Zeta potential is a powerful tool for all aspects of liposome
research
- Development of targeted drug carriers
- Coating of liposomes for longer circulation times
- Development of systems for gene therapy
- Research into the physical properties of liposomes
Optimisation of the Compacting Agent
Anionic liposomes are used to encapsulate DNA. Negatively charged DNA needs
to be condensed into small particles by adding some kind of compacting agent
such as positively charged polylysine before encapsulation. (Fig. 3)
Figure 3. Condensing plasmids to enable optimum
encapsulation in anionic liposomes.
Zeta potential measurements can be used to determine the
amount of compacting agent required to reach neutrality for high encapsulation
efficiency (Fig. 4). In addition, any subsequent modification to the surface of
the liposome can be investigated by monitoring the change in the measured zeta
potential.
Figure 4. Measurement of the zeta potential as a function
of the mole ratio of DNA:polylysine
Determination of the Charge of
Liposome/DNA Complexes
Lipofectin (Liposomal transfection agent) is a commercially available
liposome preparation for transfection. The liposomes are cationic consisting of
DOPE and DOTMA DNA is complexed with the Lipofectin. Research is going on into
the developing complexes of Lipofectin, DNA and receptorspecific peptides. Zeta
potential measurements are used to determine the charge of the resulting
complex.
Development of More Efficient
Formulations
Much work is being done in the development of vectors containing specific
molecules. Liposomal systems containing certain cell surface receptor-specific
peptides are being developed. The use of antibodies, either covalently attached
onto the surface of neutral and anionic liposomes or used in a complex, is also
being investigated.
Zeta potential measurements are being used to develop the most
efficient formulations of liposomes:peptides:DNA complexes for transfection
studies in vivo and in vitro.
Source: The Use of Zeta Potential and PCS Measurements in Gene
Therapy Research", Application Note by Malvern Instruments.
For more information on this source please visit Malvern Instruments
Ltd (UK) or Malvern Instruments
(USA).