Gene Therapy Including The Use of Zeta Potential and Photon Corellation Spectroscopy (PCS) Measurements in Gene Therapy Research With Equipment From Malvern Instruments

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).

Date Added: May 12, 2005 | Updated: Jun 11, 2013
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